Methods for detecting inflammatory bowel disease

ABSTRACT

The present invention provides for a method of detecting the presence of inflammatory bowel disease in gastrointestinal tissues or cells of a mammal by detecting increased expression of LY6 genes in the tissues or cells of the mammal relative to a control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35U.S.C. §120 to U.S. application Ser. No. 12/036,183 filed Feb. 22, 2008,now U.S. Pat. No. 7,875,431, which claims priority under 35 U.S.C.§119(e) and the benefit of U.S. Provisional Application Ser. No.60/891,196 filed Feb. 22, 2007, U.S. Provisional Application Ser. No.60/987,752 filed Nov. 13, 2007, and U.S. Provisional Application Ser.No. 61/024,170 filed Jan. 28, 2008, the entire disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to gene expression profiles ininflammatory bowel disease pathogenesis. This discovery finds use in thedetection and diagnosis of inflammatory bowel disease.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD), a chronic inflammatory disorder of thegastrointestinal tract suffered by approximately one million patients inthe United States, is made up of two major disease groups: ulcerativecolitis (UC) and Crohn's Disease (CD). In both forms of IBD, intestinalmicrobes may initiate the disease in genetically susceptibleindividuals. UC is often restricted to the colon, while CD typicallyoccurs in the ileum of the small intestine and in the colon. (Podolsky,D. K., N. Engl. J. Med. 347:417-429 (2002). Gene expression profiling oftissue from IBD patients has provided some insight into possible targetsfor therapy and/or diagnosis (see, for example, Dieckgraefe, B. K. etal., Physiol. Genomics 4:1-11 (2000); Lawrance I. C. et al., Hum MolGenet. 10:445-456 (2001); Dooley T. P. et al., Inflamm. Bowel Dis.10:1-14 (2004); and Uthoff S. M., Int J Oncol. 19:803-810 (2001)).Further investigations of gene dysregulation in patients experiencinginflammatory bowel disease include, or example, Lawrance, I. C. et al.,who disclosed distinctive gene expression profiles for several genes inUC and CD (Lawrance, I. C. et al., Human Mol. Genetics 10(5):445-456(2001)). Uthoff, S. M. S. et al. disclosed the identification ofcandidate genes for UC and CD using micro array analysis (Uthoff, S. M.S. et al., Int'l. J. Oncology 19:803-810 (2001). Dooley, T. P. et al.disclosed correlation of gene expression in IBD with drug treatment forthe disorder (Dooley, T. P. et al., Inflamm. Bowel Dis. 10(1):1-14(2004).

There is a need for the identification of additional biological markersof inflammatory bowel disease for use in diagnosis of this chronicdisease. The present disclosure fills that need.

The entire contents of all references cited herein are herebyincorporated by reference.

SUMMARY OF THE INVENTION

Disclosed herein is the unique finding that members of the LY6superfamily of genes are upregulated on the surface of intestinalepithelial cells (IEC) in models of murine colitis and in intestinaltissue of human patients experiencing IBD, which genes are not expressedon healthy IEC. The majority of LY6 family members are GPI-anchored cellsurface glycoproteins with broad distribution on cells of hematopoieticorigin, and more limited expression on non-hematopoietic cells. Thoughwidely used as markers of differentiation of immune cells (Sunderkotter,C. et al., J. Immunol. 172:4410-4417 (2004)), the functions that the LY6family possesses have been difficult to elucidate (Shevach, E. M. and P.E. Korty, Immunol. Today 10:195-200 (1989)). Reports have shown that LY6molecules are involved in a diverse array of functions including T cellactivation (Zhang, Z. X. et al., Eur. J. Immunol. 32:1584-1592 (2002)and Henderson, S. C. et al., J. Immunol. 168:118-126 (2002), olfaction(Chou, J. H. et al., Genetics 157:211-224 (2001) and cellular adhesion(Jaakkola, I. et al., J. Immunol. 170:1283-1290 (2003)).

In the broadest sense, the invention provides for a method of detectingincreased expression of genes of the human LY6 gene family in intestinaltissue in intestinal tissue from a first mammal experiencing anintestinal disorder relative to a control mammal In a more directedsense, the method is expected to be applicable to the diagnosis ofdisorders related to intestinal disorders associated with human LY6H,LYPD1, LYPD3, and LYPD5 expression, which disorders include withoutlimitation inflammatory bowel disease (IBD), such as ulcerative colitis(UC) and Crohn's Disease (CD). In one embodiment, the method of theinvention is useful to detect responders and nonresponders of IBDtherapeutic treatment. In one embodiment, the IBD is ulcerative colitis(UC). In one embodiment, the IBD is Crohn's Disease (CD). In oneembodiment, the intestinal tissue is colon tissue. In one embodiment,the colon tissue is sigmoid colon. In one embodiment, LY6H, LYPD1, LYPD3and/or LYPD5 gene expression is increased in intestinal tissue (such ascolon tissue) in an IBD, UC or CD mammal relative to normal intestinal(such as normal colon tissue) of a mammal not experiencing IBD, CD orUC. In an embodiment, the LY6H gene comprises the nucleic acid of SEQ IDNO:1 and encodes the LY6H polypeptide comprising SEQ ID NO:2. In anembodiment, the LYPD1 gene comprises the nucleic acid of SEQ ID NOS:3 or4 and encodes the LYPD1 polypeptide comprising SEQ ID NO:5. In anembodiment, the LYPD3 gene comprises the nucleic acid of SEQ ID NO:6 andencodes the LYPD3 polypeptide comprising SEQ ID NO:7. In an embodiment,the LYPD5 gene comprises the nucleic acid of SEQ ID NOS:8 or 9 andencodes the LYPD5 polypeptide comprising SEQ ID NO:10.

In one embodiment, the method of the invention comprises obtaining atissue sample from a test mammal suspected of experiencing an intestinaldisorder, contacting the tissue with a detectable agent that interactswith LY6H, LYPD1, LYPD3 and/or LYPD5 protein or with nucleic acidencoding LY6H, LYPD1, LYPD3 and/or LYPD5 and determining the level ofLY6H, LYPD1, LYPD3 and/or LYPD5 expression relative to a control tissue.In one embodiment increased expression of LY6H, LYPD1, LYPD3 and/orLYPD5 relative to control is indicative of IBD in the test mammal In oneembodiment, increased expression of LY6H, LYPD1, LYPD3 and/or LYPD5 intest intestinal tissue relative to control intestinal tissue isindicative of UC in the test mammal In one embodiment, increasedexpression of LY6H, LYPD1, LYPD3 and/or LYPD5 in test intestinal tissuerelative to control intestinal tissue is indicative of CD in the testmammal In one embodiment the tissue or cells from the test and controlmammal are from the colon.

In one embodiment, LY6H, LYPD1, LYPD3 and/or LYPD5 expression isdetermine by detection of gene expression, such as by detection of mRNAencoding LY6H, LYPD1, LYPD3 and/or LYPD5 in a tissue sample or cells. Inan embodiment, a control sample is a sample of tissue or cells of thesame tissue or cell type obtained from a mammal known not to beexperiencing a gastrointestinal disorder, such as IBD, UC or CD. In anembodiment, a control sample is a universal standard comprising RNA fromseveral normal tissues or from multiple cell lines. In microarrayanalysis, such universal standards are useful for monitoring andcontrolling intra- and inter-experimental variation. In one embodiment,a universal standard (or Universal Reference RNA (URR) is prepared asprovided in Novoradovskaya, N. et al., (2004) BMC Genomics 5:20, whichreference is hereby incorporated by reference in its entirety. In oneembodiment, for use as a control in microarray analysis of mouse RNA,the URR is a Universal Mouse Reference RNA from Stratagene® (catalog#740100, Stratagene®, La Jolla, Calif.). In one embodiment, for use as acontrol in microarray analysis of human RNA, the URR is a UniversalHuman Reference RNA from Stratagene® (catalog #740000). In oneembodiment, for use as a control in microarray analysis of rat RNA, theURR is a Universal Rat Reference RNA from Stratagene® (catalog #740200).In one embodiment, where the RNA is mouse RNA, the cell lines from whichtotal RNA is extracted comprise cell lines derived from embryo, embryofibroblast, kidney, liver hepatocyte, lung alveolar macrophage,B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast,skin, and testis. In one embodiment, where the RNA is human RNA, thecell lines from which total RNA is extracted comprise cell lines derivedfrom mammary gland adenocarcinoma, liver hepatoblastoma, cervixadenocarcinoma, embryonal carcinoma or testis, brain glioblastoma,melanoma, liposarcoma, histiocytic lymphoma (macrophage, histocyte), Tlymphoblast lymphoblastic leukemia, B lymphocyte plasmacytoma melanoma.In one embodiment where the RNA is rat RNA, the cell lines from whichtotal RNA is extracted comprise cell lines derived from blood basophilicleukemia, blood T-lymphocyte lymphoma, blood B-lymphoblast hybridoma,brain glioma, embryo yolk sac carcinoma, embryo normal fibroblast,normal kidney, liver hepatoma, lung normal alveolar macrophage, lungnormal alveolar type II, mammary gland adenocarcinoma, muscle myoblast,normal skin, and testis leydig cell tumor.

In one aspect, the invention concerns an article of manufacturecomprising a container and a composition of matter contained within thecontainer, wherein the composition of matter comprises a nucleic acidencoding LY6H, LYPD1, LYPD3 and/or LYPD5 or their complements, and/or ananti-LY6H, LYPD1, LYPD3 and/or LYPD5 antibody or antibodies, oranti-LY6H-, LYPD1-, LYPD3- and/or LYPD5-binding fragment thereof,wherein the nucleic acids and/or antiobodies are detectable. In oneembodiment, the composition of matter comprises detecting agents fordetecting nucleic acid binding, such as without limitation LY6H-,LYPD1-, LYPD3- and/or LYPD5-encoding nucleic acids or their complements,to LY6H, LYPD1, LYPD3 and/or LYPD5 nucleic acid in a tissue sample of atest mammal suspected of experiencing an intestinal disorder. In oneembodiment, the compositions of matter comprises detecting agents fordetecting antibody binding to, for example, LY6H, LYPD1, LYPD3 and/orLYPD5 in a tissue sample of a test mammal suspected of experiencing anintestinal disorder. In one embodiment, the antibody of the compositionis detectably labeled. In one embodiment, the antibody of thecomposition is detectable by a second antibody, which second antibody isdetectable or detectably labeled. The article may further optionallycomprise a label affixed to the container, or a package insert includedwith the container, that refers to the use the LY6H, LYPD1, LYPD3 and/orLYPD5 nucleic acid or its complement and/or the anti-LY6H, anti-LYPD1,anti-LYPD3 and/or anti-LYPD5 antibody or LY6H, LYPD1, LYPD3 and/or LYPD5binding fragment thereof in the detection of increased expression ofLY6H, LYPD1, LYPD3 and/or LYPD5 in intestinal tissue, including withoutlimitation, colon tissue. In an embodiment, the intestinal disorder isIBD. In an embodiment the intestinal disorder is UC or CD. In anembodiment the LYPD1 polypeptide and the anti-LYPD1 antibody is anantibody as disclosed in U.S. Pat. Nos. 7,157,558 and 7,144,990,respectively.

In one aspect, the present invention concerns a method of diagnosing thepresence of an intestinal disorder in a mammal, comprising detecting thelevel of expression of a gene encoding LY6H, LYPD1, LYPD3 and/or LYPD5polypeptide (a) in a test sample of tissue or cells obtained from saidmammal, and (b) in a control sample of known normal cells from a mammalnot experiencing an intestinal disorder of the same tissue origin ortype, wherein a higher level of expression of the LY6H, LYPD1, LYPD3and/or LYPD5 polypeptide in the test sample, as compared to the controlsample, is indicative of the presence of an intestinal disorder in themammal from which the test sample was obtained. In an embodiment, theintestinal disorder in IBD. In an embodiment, the IBD is UC. In anembodiment, the IBD is CD. In an embodiment, the detecting is bycontacting an antibody to LY6H, LYPD1, LYPD3 and/or LYPD5 polypeptide,or binding fragment of the antibody, with the test and control samplesand determining the relative amount of antibody-polypeptide complexformation. A higher level of antibody-polypeptide complex formation inthe test sample relative of the control sample is indicative ofintestinal disorder, such as IBD, UC or CD, in the test mammal Theantibody of the invention is detectably labeled or, alternatively, theantibody is detected by subsequent binding of a second antibody which isdetectable.

In yet a further embodiment, the present invention concerns a method ofdiagnosing the presence of an intestinal disorder in a mammal,comprising (a) contacting a test sample comprising tissue or cellsobtained from the test mammal with an oligonucleotide that hybridizes athigh stringency to LY6H, LYPD1, LYPD3 and/or LYPD5 nucleic acid (or itscomplement) or an antibody that binds specifically to LY6H, LYPD1, LYPD3and/or LYPD5 polypeptide and (b) detecting the formation of a complexbetween the oligonucleotide or antibody and the LY6H, LYPD1, LYPD3and/or LYPD5 nucleic acid (or its complement) or LY6H, LYPD1, LYPD3and/or LYPD5 polypeptide, respectively, in the test sample, wherein theformation of more of such complex in the test sample relative to acontrol sample is indicative of the presence of an intestinal disorder(such as IBD, UC or CD) in the test mammal In one embodiment, theintestinal disorder is IBD. In one embodiment, the disorder is UC. Inone embodiment the disorder is CD. In one embodiment the tissue of thetest and control mammals is colon tissue. Optionally, the LY6H, LYPD1,LYPD3 and/or LYPD5 polypeptide binding antibody or LY6H, LYPD1, LYPD3and/or LYPD5 gene hybridizing oligonucleotide employed by the method ofthe invention is detectable, detectably labeled, attached to a solidsupport, or the like, and/or the test sample of tissue or cells isobtained from an individual suspected of experiencing an intestinaldisorder, wherein the disorder is IBD, such as without limitation, UC orCD.

In yet a further embodiment, the present invention concerns the use of(a) a LY6H, LYPD1, LYPD3 and/or LYPD5 polypeptide, (b) a nucleic acidencoding a LY6H, LYPD1, LYPD3 and/or LYPD5 polypeptide or a vector orhost cell comprising the nucleic acid of (a), (c) an anti-LY6H, LYPD1,LYPD3 and/or LYPD5 polypeptide antibody, or (d) a LY6H, LYPD1, LYPD3and/or LYPD5-binding oligopeptide, in the preparation of a medicamentuseful for the diagnostic detection of an intestinal disorder, includingwithout limitation, IBD CD or UC, in an intestinal tissue of a mammal,including without limitation colon tissue.

In one aspect, the invention comprises a method of detecting atherapeutic drug response in a mammal treated with an IBD therapeuticagent, wherein the method comprises determining LY6H, LYPD1, LYPD3and/or LYPD5 expression in gastrointestinal tissue of a test mammalrelative to a control gastrointestinal tissue of a control mammal, wherea higher level of expression of LY6H, LYPD1, LYPD3 and/or LYPD5 in atest tissue relative to a control tissue indicates a disease state orcontinuation of the disease state. A difference in LY6H, LYPD1, LYPD3and/or LYPD5 expression in the test tissue that is not significantlyhigher than normal control expression levels or are within a range ofnormal expression levels for LY6H, LYPD1, LYPD3 and/or LYPD5 in apopulation of mammals indicates improvement or resolution of theintestinal disorder, which improvement or resolution may be attributedto the therapeutic agent. In one embodiment, a therapeutic response isdetermined when the levels of expression of LY6H, LYPD1, LYPD3 and/orLYPD5 in gastrointestinal or colon tissues or cells of the mammaltreated with a therapeutic agent are different (expression is moresimilar to normal control, i.e., LY6H, LYPD1, LYPD3 and/or LYPD5 levelsare lower than LY6H, LYPD1, LYPD3 and/or LYPD5 expression levels were inthe mammal prior to treatment.

Yet further embodiments of the present invention will be evident to theskilled artisan upon a reading of the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the nucleic acid sequence (SEQ ID NO:1) encodinghuman LY6H polypeptide and the amino acid sequence of human LY6Hpolypeptide (SEQ ID NO:2).

FIGS. 2A and 2B depict nucleic acid sequences (SEQ ID NOS:3 and 4)encoding the human LYPD1 polypeptide and the amino acid sequence ofhuman LYPD1 polypeptide shown in FIG. 2C (SEQ ID NO:5).

FIGS. 3A and 3B depict the nucleic acid sequence (SEQ ID NO:6) encodinghuman LYPD3 polypeptide and the amino acid sequence of human LYPD3polypeptide (SEQ ID NO:7).

FIGS. 4A and 4B depict nucleic acid sequences (SEQ ID NOS:8 and 9)encoding human LYPD5 polypeptide and the amino acid sequence of humanLYPD5 polypeptide shown in FIG. 4C (SEQ ID NO:10).

FIGS. 5A and 5B depict the nucleic acid sequence (SEQ ID NO:11) encodinghuman LY6D polypeptide and the amino acid sequence of human LY6Dpolypeptide (SEQ ID NO:12).

FIGS. 6A and 6B depict the nucleic acid sequence (SEQ ID NO:13) encodinghuman LY6E polypeptide and the amino acid sequence of human LY6Epolypeptide (SEQ ID NO:14).

FIGS. 7A and 7B depict the nucleic acid sequence (SEQ ID NO:15) encodinghuman LYPD2 polypeptide and the amino acid sequence of human LYPD2polypeptide (SEQ ID NO:16).

FIGS. 8A-8J depict sequences of GLG-1 (ESL-1) molecules: (A-B) AccessionNo. U64791, nucleic acid sequence (SEQ ID NO:17) encoding (C) humanGLG-1 (ESL-1) polypeptide (SEQ ID NO:18); (D-E) Accession No.NM_(—)012201, nucleic acid sequence (SEQ ID NO:19) encoding (F) humanGLG-1 (ESL-1) polypeptide (SEQ ID NO:20); (G) Accession No. AK172806,nucleic acid sequence (SEQ ID NO:21) encoding (H) human GLG-1 (ESL-1)polypeptide (SEQ ID NO:22); and (I) Accession No. AK131501, nucleic acidsequence (SEQ ID NO:23) encoding (J) human GLG-1 (ESL-1) polypeptide(SEQ ID NO:24).

FIGS. 9A and 9B depict the nucleic acid sequence (SEQ ID NO:25) encodingmurine LY6A polypeptide and the amino acid sequence of murine LY6Apolypeptide (SEQ ID NO:26).

FIGS. 10A and 10B depict the nucleic acid sequence (SEQ ID NO:27)encoding murine LY6C polypeptide and the amino acid sequence of murineLY6C polypeptide (SEQ ID NO:28).

FIGS. 11A and 11B depict the nucleic acid sequence (SEQ ID NO:29)encoding murine LY6D polypeptide and the amino acid sequence of murineLY6D polypeptide (SEQ ID NO:30).

FIGS. 12A and 12B depict the nucleic acid sequence (SEQ ID NO:31)encoding murine LY6E polypeptide and the amino acid sequence of murineLY6E polypeptide (SEQ ID NO:32).

FIGS. 13A and 13B depict the nucleic acid sequence (SEQ ID NO:33)encoding murine LY6F polypeptide and the amino acid sequence of murineLY6F polypeptide (SEQ ID NO:34).

FIGS. 14A and 14B depict the nucleic acid sequence (SEQ ID NO:35)encoding murine LY6I polypeptide and the amino acid sequence of murineLY6I polypeptide (SEQ ID NO:36).

FIGS. 15A and 15B depict the nucleic acid sequence (SEQ ID NO:37)encoding murine LY6K polypeptide and the amino acid sequence of murineLY6K polypeptide (SEQ ID NO:38).

FIGS. 16A and 16B depict the nucleic acid sequence (SEQ ID NO:45)encoding murine LYPD3 polypeptide and the amino acid sequence of murineLYPD3 polypeptide (SEQ ID NO:46).

FIGS. 17A and 17B depict the nucleic acid sequence (SEQ ID NO:47)encoding murine LY6H polypeptide and the amino acid sequence of murineLY6H polypeptide (SEQ ID NO:48).

FIGS. 18A and 18B depict the nucleic acid sequence (SEQ ID NO:49)encoding murine LYPD1 polypeptide and the amino acid sequence of murineLYPD1 polypeptide (SEQ ID NO:50).

FIGS. 19A and 19B depict the nucleic acid sequence (SEQ ID NO:51)encoding murine LYPD2 polypeptide and the amino acid sequence of murineLYPD2 polypeptide (SEQ ID NO:52).

FIGS. 20A and 20B depict the nucleic acid sequence (SEQ ID NO:53)encoding murine LY6g5c polypeptide and the amino acid sequence of murineLY6g5c polypeptide (SEQ ID NO:54).

FIGS. 21A and 22B depict the nucleic acid sequence (SEQ ID NO:55)encoding murine LY6g6c polypeptide and the amino acid sequence of murineLY6g6c polypeptide (SEQ ID NO:56).

FIGS. 22A and 22B depict the nucleic acid sequence (SEQ ID NO:57)encoding murine SLURP2/LYNX1 polypeptide and FIG. 22C depicts the aminoacid sequence of murine SLURP2/LYNX1 polypeptide (SEQ ID NO:58).

FIG. 23 shows that LY6 family members are upregulated in IEC in murinemodels of colitis. IEC in both the IL10^(−/−) (FIG. 23A) and CD45RB^(Hi)transfer colitis model (FIG. 23B) were isolated by LCM and RNA waspurified. Microarray analysis was performed and analyzed as described inthe Examples. Numbers represent the mean of the fold change compared toa universal standard RNA of colitic mice over healthy mice. Numbersbelow the heatmap indicate the inflammation score of the individualmouse.

FIGS. 24A-24D show that surface expression of LY6 molecules isupregulated on IEC in the IL10−/− model of colitis. Wild type (FIG. 24A)or IL10−/− mice (FIG. 24B) were stained for surface expression of LY6A(green, with DAPI counterstain). Similarly, wild type (FIG. 24C) orIL10−/− mice (FIG. 24D) were stained for surface expression of LY6C.

FIGS. 25A-25I show that surface expression of LY6A and LY6C areupregulated in response to inflammatory cytokines, particularly IFNγ.YAMC cells were treated with the indicated cytokine for 15 hours andstained for surface expression of LY6C (FIG. 25A) and LY6A (FIG. 25B).YAMC cells were cultured for 15 hours in the presence of increasingdoses of IFNγ and analyzed by flow cytometry for expression of LY6C(FIG. 25C) and LY6A (FIG. 25D). IFNγ stimulated YAMC cells werecollected at various time points, as indicated, and analyzed by flowcytometry for expression of LY6C (FIG. 25E) and LY6A (FIG. 25F). IL-22upregulated expression of both LY6C (FIG. 25G) and LY6A (FIG. 25H).Levels of both LY6A and LY6C were upregulated in the murine IEC line,CMT93 in response to treatment with IFNγ (FIG. 25I).

FIGS. 26A-26E Lipid raft depletion results in an inhibition ofLY6C-mediated chemokine production. Cholesterol depleted (dark bars) ornon-depleted (open bars) YAMC cells were incubated with plate-boundanti-KLH or anti-LY6C as indicated for 15 hours. RNA was collected andexpression levels of CXCL2, CXCL5, and CCL7 were determined (FIGS.26A-26C). Surface levels of LY6A (FIG. 26D) and LY6C (FIG. 26E) wheredecreased in response to cholesterol depletion.

FIGS. 27A-27D show that crosslinking of LY6C, but not LY6A, inducesupregulation of surface expression of LY6A and LY6C. YAMC cells wereincubated for 24 hours on plates coated with anti-KLH control, anti-LY6Aor anti-LY6C and analyzed by flow cytometry for expression of LY6C (FIG.27A) or LY6A (FIG. 27B). Cells were pretreated for 12 hours with 100U/ml of IFNγ and similarly plated on antibody coated plates and analyzedfor expression of LY6C (FIG. 27C) or LY6A (FIG. 27D).

FIGS. 28A-28C show that crosslinking LY6C, but not LY6A, inducessecretion of chemokines. FIG. 28A: YAMC cells were preincubated or not,as indicated, with 100 U/ml of IFNγ for 15 hours and cultured on platescoated with 10 μg/ml of anti-LY6A (black bars) or anti-LY6C (hatchedbars) or anti-KLH control (open bars). RNA was isolated at 24 (left), 48(center) and 72 (right) hours and analyzed for expression of CXCL5 orCCL7 (A). Data indicates mean±SD of the fold change (as determined by2^(−ΔΔCt) method) compared to untreated, isotype crosslinked cells. FIG.28B: Supernatants were collected at 48 hours in cells crosslinked, asabove, with 1, 5 or 10 μg/ml (as indicated) of antibody and CXCL5secretion into the supernatant was determined by ELISA. *<0.05. FIG.28C: Levels of both CXCL5 and CXCL2 in response to LY6C crosslinkingwere diminished when LY6C levels were knocked down with siRNA.

FIGS. 29A-29B show that IEC in colitis possess a similar chemokine geneexpression pattern. IEC in both the IL10^(−/−) (FIG. 29A) andCD45RB^(Hi) transfer colitis model (FIG. 29B) were isolated by LCM andRNA was purified. Microarray analysis was performed and analyzed asdescribed in the Examples. Numbers represent the mean of the fold changecompared to the universal standard RNA of colitic mice over healthymice. Numbers below the heatmap indicate the inflammation score of theindividual mouse.

FIGS. 30A-30C show that expression of human LY6 family genes isupregulated in colon cells treated with cytokines. Human Colo-205 cellswere treated with the indicated cytokines, or combinations of cytokines,for 18 or 24 hours. The fold increase in expression of human LY6H (FIG.30A), human LYPD3 (FIG. 30B), and human LYPD5 (FIG. 30C) are shownrelative to human β-actin control.

FIGS. 31A-31B show that patients with Crohn's Disease have elevatedlevels of LYPD1 (FIG. 31A) and LYPD5 (FIG. 31B) in the colon. Tissuesamples from human IBD patients were obtained and LYPD1 and LYPD5 geneexpression was determined. Statistically significant increases inexpression of LYPD1 and LYPD5 were observed in inflamed tissue of CDpatients. A statistically significant increase in expression of LYPD5was also observed in inflamed tissue of UC patients. Y-axis valuesreflect gene expression relative to a universal RNA standard.

FIGS. 32A and 32B shows (A) untransfected COS cells, and (B) COS cellstransfected with GLG-1 (ESL-1) polypeptide and stained with LYPD5-Fcprotein.

FIG. 33A depicts the structure of GLG-1 or ESL-1 and various fragmentssuitable for characterizing the binding of LYPD5 and FIG. 33B shows theresults of a co-immunoprecipitation study characterizing the binding ofLYPD5 and an LYPD5 ligand.

FIG. 34A depicts the structure of GLG-1 or ESL-1 and various fragmentssuitable for characterizing the binding of LYPD5 and FIG. 34B shows theresults of a co-immunoprecipitation study characterizing the binding ofLYPD5 and an LYPD5 ligand.

FIG. 35A depicts the structure of GLG-1 or ESL-1 and various fragmentssuitable for characterizing the binding of LYPD5 and FIG. 35B shows theresults of a co-immunoprecipitation study characterizing the binding ofLYPD5 and an LYPD5 ligand.

FIGS. 36A and 36B depict the nucleic acid sequence (SEQ ID NO: 68)encoding human integrin, beta 7, and the amino acid sequence of humanintegrin, beta 7 polypeptide (SEQ ID NO: 69).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Inflammatory Bowel Disease” or “IBD” is used interchangeably herein torefer to diseases of the bowel that cause inflammation and/or ulcerationand includes without limitation Crohn's disease and ulcerative colitis.

“Crohn's disease (CD)” or “ulcerative colitis (UC)” are chronicinflammatory bowel diseases of unknown etiology. Crohn's disease, unlikeulcerative colitis, can affect any part of the bowel. The most prominentfeature Crohn's disease is the granular, reddish-purple edmatousthickening of the bowel wall. With the development of inflammation,these granulomas often lose their circumscribed borders and integratewith the surrounding tissue. Diarrhea and obstruction of the bowel arethe predominant clinical features. As with ulcerative colitis, thecourse of Crohn's disease may be continuous or relapsing, mild orsevere, but unlike ulcerative colitis, Crohn's disease is not curable byresection of the involved segment of bowel. Most patients with Crohn'sdisease require surgery at some point, but subsequent relapse is commonand continuous medical treatment is usual.

Crohn's disease may involve any part of the alimentary tract from themouth to the anus, although typically it appears in the ileocolic,small-intestinal or colonic-anorectal regions. Histopathologically, thedisease manifests by discontinuous granulomatomas, crypt abscesses,fissures and aphthous ulcers. The inflammatory infiltrate is mixed,consisting of lymphocytes (both T and B cells), plasma cells,macrophages, and neutrophils. There is a disproportionate increase inIgM- and IgG-secreting plasma cells, macrophages and neutrophils.

Anti-inflammatory drugs sulfasalazine and 5-aminosalisylic acid (5-ASA)are useful for treating mildly active colonic Crohn's disease and iscommonly prescribed to maintain remission of the disease. Metroidazoleand ciprofloxacin are similar in efficacy to sulfasalazine and appear tobe particularly useful for treating perianal disease. In more severecases, corticosteroids are effective in treating active exacerbationsand can even maintain remission. Azathioprine and 6-mercaptopurine havealso shown success in patients who require chronic administration ofcortico steroids. It is also possible that these drugs may play a rolein the long-term prophylaxis. Unfortunately, there can be a very longdelay (up to six months) before onset of action in some patients.

Antidiarrheal drugs can also provide symptomatic relief in somepatients. Nutritional therapy or elemental diet can improve thenutritional status of patients and induce symtomatic improvement ofacute disease, but it does not induce sustained clinical remissions.Antibiotics are used in treating secondary small bowel bacterialovergrowth and in treatment of pyogenic complications.

“Ulcerative colitis (UC)” afflicts the large intestine. The course ofthe disease may be continuous or relapsing, mild or severe. The earliestlesion is an inflammatory infiltration with abscess formation at thebase of the crypts of Lieberkühn. Coalescence of these distended andruptured crypts tends to separate the overlying mucosa from its bloodsupply, leading to ulceration. Symptoms of the disease include cramping,lower abdominal pain, rectal bleeding, and frequent, loose dischargesconsisting mainly of blood, pus and mucus with scanty fecal particles. Atotal colectomy may be required for acute, severe or chronic,unremitting ulcerative colitis.

The clinical features of UC are highly variable, and the onset may beinsidious or abrupt, and may include diarrhea, tenesmus and relapsingrectal bleeding. With fulminant involvement of the entire colon, toxicmegacolon, a life-threatening emergency, may occur. Extraintestinalmanifestations include arthritis, pyoderma gangrenoum, uveitis, anderythema nodosum.

Treatment for UC includes sulfasalazine and relatedsalicylate-containing drugs for mild cases and corticosteroid drugs insevere cases. Topical adminstration of either salicylates orcorticosteroids is sometimes effective, particularly when the disease islimited to the distal bowel, and is associated with decreased sideeffects compared with systemic use. Supportive measures such asadministration of iron and antidiarrheal agents are sometimes indicated.Azathioprine, 6-mercaptopurine and methotrexate are sometimes alsoprescribed for use in refractory corticosteroid-dependent cases.

As used herein, “LY6 gene family member” or “LY6 gene superfamilymember” is used interchangeably herein to refer to a gene havinghomology to members of the LY6 gene family, the majority of which genefamily members are GPI-anchored cell surface glycoproteins with broaddistribution on cells of hematopoietic origin and more limitedexpression on non-hematopoietic cells. Members of this gene family areused as markers of differentiation of immune cells (Sunderkotter, C. etal., J. Immunol. 172:4410-4417 (2004)). Genes of the LY6 family havebeen examined (Shevach, E. M. and P. E. Korty, Immunol. Today 10:195-200(1989)) and functions include T cell activation (Zhang, Z. X. et al.,Eur. J. Immunol. 32:1584-1592 (2002) and Henderson, S. C. et al., J.Immunol. 168:118-126 (2002), olfaction (Chou, J. H. et al., Genetics157:211-224 (2001) and cellular adhesion (Jaakkola, I. et al., J.Immunol. 170:1283-1290 (2003)). Members of the LY6 gene family includewithout limitation members of the mammalian LY6 gene family, such as theLY6 family genes of mouse or human. As use here, “LY6 gene” refers to aLY6 gene family member and “LY6 polypeptide” refers to the polypeptideencoded by a LY6 gene. Murine LY6 gene family members include, withoutlimitation, LY6A (NM_(—)010738, nucleic acid SEQ ID NO:25 which encodespolypeptide SEQ ID NO:26), LY6C (NM_(—)010741, nucleic acid SEQ ID NO:27which encodes polypeptide SEQ ID NO:28), LY6D (NM_(—)003695, nucleicacid SEQ ID NO:29 which encodes polypeptide SEQ ID NO:30), LY6E(NM_(—)002346, nucleic acid SEQ ID NO:31 which encodes polypeptide SEQID NO:32), LY6F (NM_(—)008530, nucleic acid SEQ ID NO:33 which encodespolypeptide SEQ ID NO:34), LY6I (NM_(—)020498, nucleic acid SEQ ID NO:35which encodes polypeptide SEQ ID NO:36), and LY6K (NM_(—)017527, nucleicacid SEQ ID NO:37 which encodes polypeptide SEQ ID NO:38). Human LY6gene family members include, without limitation, LY6H (NM_(—)002347,nucleic acid SEQ ID NO:1 which encodes polypeptide SEQ ID NO:2), LYPD1(NM_(—)144586, nucleic acid SEQ ID NOS:3 or 4 which encodes polypeptideSEQ ID NO:5), LYPD3 (NM_(—)014400, nucleic acid SEQ ID NO:6 whichencodes polypeptide SEQ ID NO:7), LYPD5 (NM_(—)182573, nucleic acid SEQID NOS:8 or 9 which encodes polypeptide SEQ ID NO:10), LY6D(NM_(—)003695, nucleic acid SEQ ID NO:11 which encodes polypeptide SEQID NO:12), LY6E (NMNM_(—)002346, nucleic acid SEQ ID NO:13 which encodespolypeptide SEQ ID NO:14), LYPD2 (NM_(—)205545, nucleic acid SEQ IDNO:15 which encodes polypeptide SEQ ID NO:16). In embodiments, thepolynucleotide of each LY6 gene family member disclosed herein comprisesat least 15, at least 25, at least, at least 50, at least 100, at least250, at least 500, at least 750, at least 1000, at least 1250, at least1500, at least 1750, at least 2000, or at least 2040 contiguousnucleotides of SEQ ID NOs:1, 3, 4, 6, 8, 9, 11, 13, 15, 25, 27, 29, 31,33, 35, 37, 45, 47, 49, 51, 53, 55, or 57, or the LY6 gene family memberpolynucleotide comprises SEQ ID NOS:1, 3, 4, 6, 8, 9, 11, 13, 15, 25,27, 29, 31, 33, 35, 37, 45, 47, 49, 51, 53, 55, or 57. In oneembodiment, a polynucleotide that binds a LY6 gene family memberpolynucleotide (SEQ ID NOs:1, 3, 4, 6, 8, 9, 11, 13, 15, 25, 27, 29, 31,33, 35, 37, 45, 47, 49, 51, 53, 55, or 57), or fragment thereof, has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with the LY6polypeptide or fragment thereof. In one embodiment, the LY6 gene familymember polypeptide comprises at least 10, at least 25, at least 50, atleast 75, at least 100, at least 125, at least 150, at least 175, atleast 200, at least 225, at least 250, at least 275, at least 300, or atleast 325, at least contiguous amino acids of SEQ ID NOs:2, 5, 7, 10,12, 14, 26, 28, 30, 32, 34, 36, 38, 46, 48, 50, 52, 54, 56, or 58, orthe LY6 gene family polypeptide comprises SEQ ID NOs:2, 5, 7, 10, 12,14, 26, 28, 30, 32, 34, 36, 38, 46, 48, 50, 52, 54, 56, or 58).

A “native sequence polypeptide” of any of the LY6 gene family memberscomprises a polypeptide having the same amino acid sequence as thecorresponding LY6 gene family member polypeptide derived from nature.Such native sequence LY6 polypeptides can be isolated from nature or canbe produced by recombinant or synthetic means. The term “native sequenceLY6 polypeptide” specifically encompasses naturally-occurring truncatedor secreted forms of the specific LY6 polypeptide (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe polypeptide. In one specific aspect, the native sequence LY6polypeptides disclosed herein are mature or full-length native sequencepolypeptides corresponding to the sequences in FIGS. 1-7 and SEQ IDNOs:2, 5, 7, 10, 12, 14, 26, 28, 30, 32, 34, 36, 38, 46, 48, 50, 52, 54,56, or 58.

As used herein, a “LY6 polypeptide variant” means a LY6 polypeptide,preferably biologically active forms thereof, as defined herein, havingat least about 80% amino acid sequence identity with a full-lengthnative sequence LY6 polypeptide sequence, as disclosed herein, andvariant forms thereof lacking the signal peptide, an extracellulardomain, or any other fragment of a full length native sequence LY6polypeptide such as those referenced herein. Such variant polypeptidesinclude, for instance, polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- or C-terminus of thefull-length native amino acid sequence. In a specific aspect, suchvariant polypeptides will have at least about 80% amino acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% aminoacid sequence identity, to a full-length native sequence LY6 polypeptidesequence polypeptide, as disclosed herein, and variant forms thereoflacking a signal peptide, an extracellular domain, or any other fragmentof a full length native sequence LY6 polypeptide such as those disclosedherein.

“Percent (%) amino acid sequence identity” with respect to a LY6polypeptide sequence identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific LY6 polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

As used herein “LY6 variant polynucleotide” or “LY6 variant nucleic acidsequence,” or “LY6 gene” refers to a nucleic acid molecule which encodesa LY6 gene family member polypeptide, preferably biologically activeforms thereof, as defined herein, and which have at least about 80%nucleic acid sequence identity with a nucleotide acid sequence encodinga full-length native sequence LY6 polypeptide sequence identifiedherein, or any other fragment of the respective full-length LY6polypeptide sequence as identified herein (such as those encoded by anucleic acid that represents only a portion of the complete codingsequence for a full-length LY6 polypeptide). Ordinarily, such variantpolynucleotides will have at least about 80% nucleic acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%nucleic acid sequence identity with a nucleic acid sequence encoding therespective full-length native sequence LY6 polypeptide sequence or anyother fragment of the respective full-length LY6 polypeptide sequenceidentified herein. Such variant polynucleotides do not encompass thenative nucleotide sequence.

Ordinarily, such variant polynucleotides vary at least about 50nucleotides in length from the native sequence polypeptide,alternatively the variance can be at least about 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length.

“Percent (%) nucleic acid sequence identity” with respect to a LY6 genepolypeptide-encoding nucleic acid sequences identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with the nucleotides in the LY6 gene nucleic acid sequence ofinterest, respectively, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposesherein, however, % nucleic acid sequence identity values are generatedusing the sequence comparison computer program ALIGN-2, wherein thecomplete source code for the ALIGN-2 program is provided in Table 1below. The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in Table 1 below has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. or may be compiled from thesource code provided in Table 1 below. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program=s alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “REF-DNA”,wherein “REF-DNA” represents a hypothetical LY6 gene-encoding nucleicacid sequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “REF-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides. Unless specifically statedotherwise, all % nucleic acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

In other embodiments, LY6 gene variant polynucleotides are nucleic acidmolecules that encode LY6 polypeptide, respectively, and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length LY6polypeptide, respectively, as disclosed herein. Such variantpolypeptides may be those that are encoded by such variantpolynucleotides.

“Isolated”, when used to describe the various LY6 polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, such polypeptideswill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Such isolated polypeptides includes the correspondingpolypeptides in situ within recombinant cells, since at least onecomponent of the LY6 polypeptide from its natural environment will notbe present. Ordinarily, however, such isolated polypeptides will beprepared by at least one purification step.

An “isolated” LY6 polypeptide-encoding nucleic acid is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the polypeptide-encoding nucleic acid. Any of theabove such isolated nucleic acid molecule is other than in the form orsetting in which it is found in nature. Any such nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein “expression” as applied to gene expression, refers totranscription of a gene encoding a protein to produce mRNA as well astranslation of the mRNA to produce the protein encoded by the gene.Thus, increased or decreased expression refers to increased or decreasedtranscription of a gene and/or increased or decreased translation ofmRNA resulting from transcription.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50EC;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42EC; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5× Denhardt=s solution, sonicated salmonsperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42EC, with a10 minute wash at 42EC in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55EC.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37EC in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt=s solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50EC. The ordinarily skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising an LY6 polypeptide, or LY6 polypeptide bindingagent fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with the activity ofthe polypeptide to which it is fused. The tag polypeptide preferablyalso is sufficiently unique so that such antibody does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least six amino acid residues and usually between about 8 and 50amino acid residues (preferably, between about 10 and 20 amino acidresidues).

“Active” or “activity” for the purposes herein refers to form(s) ofpolypeptides which retain a biological and/or an immunological activityof native or naturally-occurring polypeptide, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring polypeptide otherthan the ability to induce the production of an antibody against anantigenic epitope possessed by a native or naturally-occurringpolypeptide, and an “immunological” activity refers to the ability toinduce the production of an antibody against an antigenic epitopepossessed by a native or naturally-occurring polypeptide. An activepolypeptide, as used herein, is an antigen that is differentiallyexpressed, either from a qualitative or quantitative perspective, in IBDtissue, relative to its expression on similar tissue that is notafflicted with IBD.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide disclosed herein. Suitableantagonist molecules specifically include antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying antagonists may comprisecontacting such a polypeptide, including a cell expressing it, with acandidate agonist or antagonist molecule and measuring a detectablechange in one or more biological activities normally associated withsuch polypeptide.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the progression of a disease.Treatment may also refer the modification of the progression of an IBD.

“Diagnosing” refers to the process of identifying or determining thedistinguishing characteristics of a disease including without limitationIBD, UC and/or Crohn's Disease. The process of diagnosing is sometimesalso expressed as staging or disease classification based on severity ordisease progression as well as on location (such as, for example,location within or along the gastrointestinal tract at whichinflammation and/or altered gene expression is found).

Subjects in need of diagnosis include those already experiencing withaberrant LY6 expression as well as those prone to having or those inwhom aberrant LY6 expression is to be prevented. Accordingly, an aspectof the invention is the detection of a therapeutic drug response in amammal treated with a therapeutic agent for the treatment of IBD,wherein the method comprises determining Ih LY6 expression ingastrointestinal tissue of a test mammal relative to a control anddetermining that the LY6 expression levels are within not significantlydifferent from normal control expression levels. In one embodiment, atherapeutic response is determined when the levels of expression of LY6of the mammal treated with a therapeutic agent are different (expressionis more similar to normal control, i.e., LY6 expression levels are lowerthan LY6 expression levels were in the mammal prior to treatment).

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For IBD therapy, efficacy can be measured, for example, byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR). Biopsies may be taken to assess gene expression andobserve histopathology of gastrointestinal tissue from the patient. Theinvention described herein relating to the process of prognosing and/ordiagnosing involves the determination and evaluation of LY6 geneexpression upregulation.

“Mammal” or “mammalian subject” for purposes of the treatment of,alleviating the symptoms of or diagnosis of a IBD refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, ferrets, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN7, polyethylene glycol (PEG), and PLURONICS7.

By “solid phase” or “solid support” is meant a non-aqueous matrix towhich a polypeptide, nucleic acid, antibody or LY6 binding agent canadhere or attach. Examples of solid phases encompassed herein includethose formed partially or entirely of glass (e.g., controlled poreglass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,polyvinyl alcohol and silicones. In certain embodiments, depending onthe context, the solid phase can comprise the well of an assay plate; inothers it is a purification column (e.g., an affinity chromatographycolumn) This term also includes a discontinuous solid phase of discreteparticles, such as those described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drugto a mammal The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

A “small molecule” or “small organic molecule” is defined herein to havea molecular weight below about 500 Daltons.

An “effective amount” of an antagonist agent is an amount sufficient tobring about a physiological effect, such as without limitation toinhibit, partially or entirely, function of gene or its encoded protein.An “effective amount” may be determined empirically and in a routinemanner, in relation to this purpose.

The term “therapeutically effective amount” refers to an antagonist orother drug effective to “treat” a disease or disorder in a subject ormammal In the case of IBD, the therapeutically effective amount of thedrug will restore aberrant LY6 expression to normal physiologicallevels; reduce gastrointestinal inflammation; reduce the number ofgastrointestinal lesions; and/or relieve to some extent one or more ofthe symptoms associated with IBD, UC and/or CD. See the definitionherein of “treating”.

A “growth inhibitory amount” of an antagonist is an amount capable ofinhibiting the growth of a cell, especially tumor, e.g., cancer cell,either in vitro or in vivo. For purposes of inhibiting neoplastic cellgrowth, such an amount may be determined empirically and in a routinemanner.

A “cytotoxic amount” of an antagonist is an amount capable of causingthe destruction of a cell, especially a proliferating cell, e.g., cancercell, either in vitro or in vivo. For purposes of inhibiting neoplasticcell growth may be determined empirically and in a routine manner.

The term “antibody” is used in the broadest sense and specificallycovers, for example, anti-LY6 monoclonal antibodies (includingantagonist and neutralizing antibodies), anti-LY6 antibody compositionswith polyepitopic specificity, polyclonal antibodies, single chainanti-LY6 antibodies, multispecific antibodies (e.g., bispecific) andantigen binding fragments (see below) of all of the above enumeratedantibodies as long as they exhibit the desired biological orimmunological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with antibody herein.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the approximately 110-amino acid span of thevariable domains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around about Kabatresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout Kabat residues 31-35B (H1), 50-65 (H2) and 95-102 (H3) in theV_(H) (Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.around about Chothia residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) inthe V_(L), and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in the V_(H)(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical and/orbind the same epitope(s), except for possible variants that may ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Such monoclonal antibody typicallyincludes an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones or recombinant DNA clones. It should be understood that theselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity, themonoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler et al.,Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier,N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567), phage display technologies (see, e.g., Clackson et al.,Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597(1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al.,J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci.USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669(all of GenPharm); U.S. Pat. No. 5,545,807; WO 1997/17852; U.S. Pat.Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016; Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg etal., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994);Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger,Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern.Rev. Immunol., 13: 65-93 (1995).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient or acceptor antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-human species (donor antibody) such as mouse, rat,rabbit or nonhuman primate having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance such asbinding affinity. Generally, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence although theFR regions may include one or more amino acid substitutions that improvebinding affinity. The number of these amino acid substitutions in the FRare typically no more than 6 in the H chain, and in the L chain, no morethan 3. The humanized antibody optionally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (VH), and the first constant domain of one heavychain (C_(H)1). Each Fab fragment is monovalent with respect to antigenbinding, i.e., it has a single antigen-binding site. Pepsin treatment ofan antibody yields a single large F(ab′)₂ fragment which roughlycorresponds to two disulfide linked Fab fragments having divalentantigen-binding activity and is still capable of cross-linking antigen.Fab=fragments differ from Fab fragments by having additional fewresidues at the carboxy terminus of the C_(H)1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

As used herein “LY6 binding polypeptide” is an oligopeptide that binds,preferably specifically, to a LY6 polypeptide, ligand or signalingcomponent, respectively, or a LY6 binding portion or fragment thereof.Such oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Such oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more. Such oligopeptides may be identifiedwithout undue experimentation using well known techniques. In thisregard, it is noted that techniques for screening oligopeptide librariesfor oligopeptides that are capable of specifically binding to apolypeptide target are well known in the art (see, e.g., U.S. Pat. Nos.5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564;Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysenet al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen etal., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. Proc. Natl. Acad. Sci. USA,87:6378 (1990); Lowman, H. B. et al. Biochemistry, 30:10832 (1991);Clackson, T. et al. Nature, 352: 624 (1991); Marks, J. D. et al., J.Mol. Biol., 222:581 (1991); Kang, A. S. et al. Proc. Natl. Acad. Sci.USA, 88:8363 (1991), and Smith, G. P., Current Opin. Biotechnol., 2:668(1991).

An LY6 antagonist (e.g., antibody, polypeptide, oligopeptide or smallmolecule) “which binds” a target antigen of interest, e.g. LY6 is onethat binds the target with sufficient affinity so as to be a usefuldiagnostic, prognostic and/or therapeutic agent in targeting a cell ortissue expressing the antigen, and does not significantly cross-reactwith other proteins. The extent of binding to a non-desired markerpolypeptide will be less than about 10% of the binding to the particulardesired target, as determinable by common techniques such asfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

Moreover, the term “specific binding” or “specifically binds to” or is“specific for” a particular LY6 polypeptide or an epitope on aparticular LY6 polypeptide target means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target, for example, an excess of non-labeled target. Inthis case, specific binding is indicated if the binding of the labeledtarget to a probe is competitively inhibited by excess unlabeled target.In one embodiment, such terms refer to binding where a molecule binds toa particular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.Alternatively, such terms can be described by a molecule having a Kd forthe target of at least about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M,10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or greater.

A gastrointestinal cell or tissue that “overexpresses” LY6 if that cellor tissue is shown to have increased nucleic acid encoding LY6 in acells or if that cell or tissue over produces and secretes LY6 protein,compared to a normal gastrointestinal cell or tissue of the same tissuetype. Such overexpression may result from gene amplification or byincreased transcription or translation. Various diagnostic or prognosticassays are known that measure altered expression levels resulting inincreased or decreased levels at the cell surface or increased ordecreased levels of secreted protein and include without limitationimmunohistochemistry assay using anti-LY6 antibodies, FACS analysis,etc. Alternatively, the levels of LY6 encoding nucleic acid or mRNA canbe measured in the cell, e.g., via fluorescent in situ hybridizationusing a nucleic acid based probe corresponding to a LY6-encoding nucleicacid or the complement thereof; (FISH; see WO98/45479 published October,1998), Southern blotting, Northern blotting, or polymerase chainreaction (PCR) techniques, such as real time quantitative PCR (RT-PCR).Alternatively, LY6 polypeptide overexpression is determinable bymeasuring shed antigen in a biological fluid such as serum, e.g, usingantibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issuedJun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No.5,401,638 issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods132:73-80 (1990)). In addition to the above assays, various in vivoassays are available to the skilled practitioner. For example, one mayexpose cells within the body of the patient to an antibody which isoptionally labeled with a detectable label, e.g., a radioactive isotope,and binding of the antibody to cells in the patient can be evaluated,e.g., by external scanning for radioactivity or by analyzing a biopsytaken from a patient previously exposed to the therapeutic agent.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody,oligopeptide or other organic molecule so as to generate a “labeled”antibody, oligopeptide or other organic molecule. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, enzymes and fragments thereofsuch as nucleolytic enzymes, antibiotics, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof, andthe various antitumor or anticancer agents disclosed below. Othercytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “chemotherapeutic agent” or “therapeutic agent” is a chemical compounduseful in the treatment of a disorder or disease. Examples ofchemotherapeutic or therapeutic agents for the treatment of IBD includewithout limitation anti-inflammatory drugs sulfasalazine and5-aminosalisylic acid (5-ASA); metroidazole and ciprofloxacin aresimilar in efficacy to sulfasalazine and appear to be particularlyuseful for treating perianal disease; in more severe cases,corticosteroids are effective in treating active exacerbations and caneven maintain remission; azathioprine, 6-mercaptopurine, andmethotrexate have also shown success in patients who require chronicadministration of cortico steroids; antidiarrheal drugs can also providesymptomatic relief in some patients; nutritional therapy or elementaldiet can improve the nutritional status of patients and inducesymtomatic improvement of acute disease; antibiotics are used intreating secondary small bowel bacterial overgrowth and in treatment ofpyogenic complications. IBD chemotherapeutic agents further includebiologicals and other agents as follows: anti-beta7 antibodies (see, forexample, WO2006026759), anti-alpha4 antibodies (such as ANTEGEN®),anti-TNF antibody (REMICADE®)) or non-protein compounds includingwithout limitation 5-ASA compounds ASACOL®, PENTASA™, ROWASA™, COLAZAL™,and other compounds such as Purinethol and steroids such as prednisone.Examples of chemotherapeutic agents for the treatment of cancer includehydroxyureataxanes (such as paclitaxel and doxetaxel) and/oranthracycline antibiotics; alkylating agents such as thiotepa andCYTOXAN7 cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL7); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN7), CPT-11 (irinotecan, CAMPTOSAR7), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN 7 doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK7 polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE7, FILDESIN7); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL7 paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE7 doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR7); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN7); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN7); oxaliplatin; leucovovin; vinorelbine(NAVELBINE7); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA7);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon -α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

“Epithelia,” “epithelial” and “epithelium” refer to the cellularcovering of internal and external body surfaces (cutaneous, mucous andserous), including the glands and other structures derived therefrom,e.g., corneal, esophageal, epidermal, and hair follicle epithelialcells. Other exemplary epithelial tissue includes: olfactoryepithelium—the pseudostratified epithelium lining the olfactory regionof the nasal cavity, and containing the receptors for the sense ofsmell; glandular epithelium—the epithelium composed of secreting cellssquamous epithelium; squamous epithelium—the epithelium comprising oneor more cell layers, the most superficial of which is comosed of flat,scalelike or platelike cells. Epithelium can also refer to transitionalepithelium, like that which is characteristically found lining holloworgans that are subject to great mechanical change due to contractionand distention, e.g., tissue which represents a transition betweenstratified squamous and columnar epithelium.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “LY6” or “LY6 polypeptide” is used herein to refer genericallyto any of the mammalian homologs of the mammalian LY6 gene family. Theterm “LY6” may be used to describe protein or nucleic acid.

The term “overexpression” as used herein, refers to cellular geneexpression levels of a tissue that is higher than the normal expressionlevels for that tissue. The term “underexpression” as used herein,refers to cellular gene expression levels of a tissue that is lower thanthe normal expression levels for that tissue. In either case, the higheror lower expression is significantly different from normal expressionunder controlled conditions of the study.

A “control” includes a sample obtained for use in determining base-lineor normal expression or activity in a mammal that is not experiencingIBD. Accordingly, a control sample may be obtained by a number of meansincluding from tissue or cells not affected by inflammation and/or IBD,UC or CD (as determined by standard techniques); non-IBD cells or tissuee.g., from cells of a subject not experiencing IBD; from subjects nothaving an IBD, Crohn's disease, or ulcerative colitis disorder; fromsubjects not suspected of being at risk for an IBD, CD or UC; or fromcells or cell lines derived from such subjects. A control also includesa previously established standard. For assays, such as mRNA assays,including microarray assays, a control may be a universal control. Suchuniversal control refers to RNA expression information of a particularLY6 gene obtained from RNA isolated from a mixture of healthy tissues orfrom a mixture of cell lines derived from various tissues such as,without limitation, universal reference RNAs disclosed herein.Accordingly, any test or assay conducted according to the invention maybe compared with the established standard and it may not be necessary toobtain a control sample for comparison each time.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M  −8  /* value of a match with a stop */int   _day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V WX Y Z */ /* A */ { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2,1, 1, 0, 0,−6, 0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0,0,−3,−2, 2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */{−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */ { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */{−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */ {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */{−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */{−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */ {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */ { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */ { 0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };/*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* maxjumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gapslarger than this */ #define JMPS 1024 /* max jmps in an path */ #defineMX 4 /* save if there's at least MX−1 bases since last jmp */ #defineDMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty formismatched bases */ #define DINS0 8 /* penalty for a gap */ #defineDINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP];/* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no.of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 −1 */struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for errmsgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag:nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’-‘A’))|(1<<(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’-‘A’))|(1<<(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\”align.out\“\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */id = xx − yy + len1 − 1; ...nw if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy); if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx< len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax= col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void)free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print( ) -- only routinevisible outside this module  *  * static:  * getmat( ) -- trace backbest path, count matches: print( )  * pr_align( ) -- print alignment ofdescribed in array p[ ]: print( )  * dumpblock( ) -- dump a block oflines with numbers, stars: pr_align( )  * nums( ) -- put out a numberline: dumpblock( )  * putline( ) -- put out a line (name, [num], seq,[num]): dumpblock( )  * stars( ) - -put a line of stars: dumpblock( )  *stripname( ) -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC 3 #define P_LINE 256 /* maximum output line */#define P_SPC 3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print( ) print { int lx, ly, firstgap, lastgap; /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr,“%s: can't write %s\n”,prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s (length =%d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s (length =%d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1; firstgap =lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */ pp[0].spc =firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if (dmax > len1 − 1){ /* leading gap in y */ pp[1].spc = firstgap = dmax − (len1 − 1); lx −=pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gap in x */ lastgap =len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 > len0 − 1) { /*trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −= lastgap; }getmat(lx, ly, firstgap, lastgap); pr_align( ); } /*  * trace back thebest path, count matches  */ static getmat(lx, ly, firstgap, lastgap)getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap, lastgap;/* leading trailing overlap */ { int nm, i0, i1, siz0, siz1; charoutx[32]; double pct; register n0, n1; register char *p0, *p1; /* gettotal matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “”:“s”); fprintf(fx,“%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy ==1)? “”:“s”); fprintf(fx,“%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars( ) */ /*  * print alignment of described in struct path pp[ ] */ static pr_align( ) pr_align { int nn; /* char count */ int more;register i; for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /*  *do we have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) { /* leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if(siz[i]) { /* in a gap */ *po[i]++ = ‘-’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register i; for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) !=‘ ’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( );putline(i); if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1)nums(i); } } } /*  * put out a number line: dumpblock( )  */ staticnums(ix) nums int ix; /* index in out[ ] holding seq line */ { charnline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn =nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py == ‘ ’ || *py == ‘-’) *pn = ‘ ’;else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i;for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘-’;} else *pn = ‘ ’; i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn;pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out aline (name, [num], seq, [num]): dumpblock( )  */ static putline(ix)putline int ix; { ...putline int i; register char *px; for (px =namex[ix], i = 0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx);for (; i < lmax+P_SPC; i++) (void) putc(‘ ’, fx); /* these count from 1: * ni[ ] is current element (from 1)  * nc[ ] is number at start ofcurrent line  */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx); (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock( )  */ static stars( ) stars { int i;register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ‘ ’ &&*(po[0]) == ‘ ’) ||  !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’))return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for (p0 =out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,“r”)) == 0) {fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s: malloc( ) failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } /*  *write a filled jmp struct offset of the prev one (if any): nw( )  */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 Reference XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the referencepolypeptide) = 5 divided by 15 = 33.3%

TABLE 3 Reference XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the referencepolypeptide) = 5 divided by 10 = 50%

TABLE 4 Reference- NNNNNNNNNNNNNN (Length = 14 nucleotides) DNAComparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acidsequence identity = (the number of identically matching nucleotidesbetween the two nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the reference-DNA nucleic acidsequence) = 6 divided by 14 = 42.9%

TABLE 5 Reference-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the reference-DNA nucleic acid sequence) = 4divided by 12 = 33.3%Diagnostic Methods of the Invention

It is further contemplated that use of therapeutic agents for IBD may bespecifically targeted to disorders where the affected tissue and/orcells exhibit increased LY6 expression relative to control. Accordingly,it is contemplated that the detection of increased LY6 expression may beused to detect IBD, such as CD or UC, in the gastrointestinal tissue ofa mammal and/or to identify tissues and disorders that will particularlybenefit from treatment with an IBD therapeutic agent, including achemotherapeutic agent, useful in ameliorating IBD, UC and/or CD in ahuman patient.

In preferred embodiments, LY6 expression levels are detected, either bydirect detection of the gene transcript or by detection of proteinlevels or activity. Transcripts may be detected using any of a widerange of techniques that depend primarily on hybrization or probes tothe LY6 mRNA transcripts, to cDNAs synthesized therefrom, or to DNAwhere LY6 gene amplification is present. Well known techniques includeNorthern blotting, reverse-transcriptase PCR and microarray analysis oftranscript levels. Methods for detecting LY6 protein levels includeWestern blotting, immunoprecipitation, two-dimensional polyacrylatmidegel electrophoresis (2D SDS-PAGE—preferably compared against a standardwherein the position of the LY6 proteins has been determined), and massspectroscopy. Mass spectroscopy may be coupled with a series ofpurification steps to allow high-throughput indentification of manydifferent protein levels in a particular sample. Mass spectroscopy and2D SDS-PAGE can also be used to identify post-transcriptionalmodifications to proteins including proteolytic events, ubiquitination,phosphorylation, lipid modification, etc. LY6 activity may also beassessed by analyzing binding to substrate DNA or in vitrotranscriptional activiaton of target promoters. Gel shift assay, DNAfootprinting assays and DNA-protein crosslinking assays are all methodsthat may be used to assess the presence of a protein capable of bindingto Gli binding sites on DNA. J Mol. Med 77(6):459-68 (1999); Cell100(4): 423-34 (2000); Development 127(19): 4923-4301 (2000).

In certain embodiments, LY6 transcript levels are measured, and diseasedor disordered tissues showing significantly elevated LY6 levels relativeto control are treated with an IBD therapeutic compound. Accordingly,LY6 expression levels are a powerful diagnostic measure for determiningwhether a patient is experiencing IBD and whether that patient shouldreceive an IBD therapeutic agent.

Antibody Compositions for Use in the Methods of the Invention

A. Anti-LY6 Antibodies

In one embodiment, the present invention provides the use of anti-LY6antibodies, which may find use herein as therapeutic, diagnostic and/orprognostic agents in determining the existence, severity of and/orprognosing the disease course of an inflammatory bowel disease such asUC. Exemplary antibodies that may be used for such purposes includepolyclonal, monoclonal, humanized, bispecific, and heteroconjugateantibodies. The term “antibodies” sometimes also include antigen-bindingfragments. Anti-LY6 antibodies are available commercially, such as forexample, from R&D Systems, Minneapolis, Minn. Antiobodies that bindspecifically to LY6 as antigen may be obtained commercially or preparedby standard methods known in the art of antibody and protein chemistryfor use in the method of the invention. Antiobodies to LYPD1 aredisclosed, for example in U.S. Pat. No. 7,144,990, the disclosure patentis hereby incorporated by reference in its entirety.

1. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g, by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-LY6 antibodies useful in the practice of the invention mayfurther comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of a humanized anti-LY6 antibody antibodies arecontemplated. For example, the humanized antibody may be an antibodyfragment, such as a Fab, which is optionally conjugated with one or morecytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized antibody may be an intact antibody, such asan intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, while retaining similar antigenbinding specificity of the corresponding full length molecule, and maylead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and scFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind separate antigens or bind to two different epitopes of a particularLY6 polypeptide described herein. Other such antibodies may combine theabove LY6 binding site with a binding site for another protein. Wherethe bispecific antibody is useful in the diagnostic method of theinvention, the second antibody arm may bind a detectable polypeptide.Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies alsofind use in the present method of the invention by providing multiple(either different or the same) detectable markers on each antibody forimproved assay detection. Heteroconjugate antibodies may be made usingany convenient cross-linking methods. Suitable cross-linking agents arewell known in the art, and are disclosed in U.S. Pat. No. 4,676,980,along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

7. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

8. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, agrowth inhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate) and/or a detectable label.

a. Chemotherapeutic Agents

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

B. LY6 Binding Oligopeptides

LY6 binding oligopeptides of the present invention are oligopeptidesthat bind, preferably specifically, to a LY6 polypeptide as describedherein. LY6 binding oligopeptides may be chemically synthesized usingknown oligopeptide synthesis methodology or may be prepared and purifiedusing recombinant technology. LY6 binding oligopeptides are usually atleast about 5 amino acids in length, alternatively at least about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acids in length or more, wherein such oligopeptidesthat are capable of binding, preferably specifically, to a LY6polypeptide as described herein. LY6 binding oligopeptides may beidentified without undue experimentation using well known techniques. Inthis regard, it is noted that techniques for screening oligopeptidelibraries for oligopeptides that are capable of specifically binding toa polypeptide target are well known in the art (see, e.g., U.S. Pat.Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564;Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysenet al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen etal., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci.USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991),J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

In this regard, bacteriophage (phage) display is one well knowntechnique which allows one to screen large oligopeptide libraries toidentify member(s) of those libraries which are capable of specificallybinding to a polypeptide target. Phage display is a technique by whichvariant polypeptides are displayed as fusion proteins to the coatprotein on the surface of bacteriophage particles (Scott, J. K. andSmith, G. P. (1990) Science 249: 386). The utility of phage display liesin the fact that large libraries of selectively randomized proteinvariants (or randomly cloned cDNAs) can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl.Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have beenused for screening millions of polypeptides or oligopeptides for oneswith specific binding properties (Smith, G. P. (1991) Current Opin.Biotechnol., 2:668). Sorting phage libraries of random mutants requiresa strategy for constructing and propagating a large number of variants,a procedure for affinity purification using the target receptor, and ameans of evaluating the results of binding enrichments. U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,689, and 5,663,143.

Although most phage display methods have used filamentous phage,lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024),T4 phage display systems (Ren et al., Gene, 215: 439 (1998); Zhu et al.,Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection &Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311(1997); Ren, Protein Sci., 5: 1833 (1996); Efimov et al., Virus Genes,10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methodsin Enzymology, 217: 228-257 (1993); U.S. Pat. No. 5,766,905) are alsoknown.

Many other improvements and variations of the basic phage displayconcept have now been developed. These improvements enhance the abilityof display systems to screen peptide libraries for binding to selectedtarget molecules and to display functional proteins with the potentialof screening these proteins for desired properties. Combinatorialreaction devices for phage display reactions have been developed (WO98/14277) and phage display libraries have been used to analyze andcontrol bimolecular interactions (WO 98/20169; WO 98/20159) andproperties of constrained helical peptides (WO 98/20036). WO 97/35196describes a method of isolating an affinity ligand in which a phagedisplay library is contacted with one solution in which the ligand willbind to a target molecule and a second solution in which the affinityligand will not bind to the target molecule, to selectively isolatebinding ligands. WO 97/46251 describes a method of biopanning a randomphage display library with an affinity purified antibody and thenisolating binding phage, followed by a micropanning process usingmicroplate wells to isolate high affinity binding phage. The use ofStaphlylococcus aureus protein A as an affinity tag has also beenreported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describesthe use of substrate subtraction libraries to distinguish enzymespecificities using a combinatorial library which may be a phage displaylibrary. A method for selecting enzymes suitable for use in detergentsusing phage display is described in WO 97/09446. Additional methods ofselecting specific binding proteins are described in U.S. Pat. Nos.5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these librariesare also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717,5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and5,723,323.

In aspect, the present invention concerns ligands for the LYPD5polypeptide. FIG. 32 demonstrates this showing untransfected COS cells(A) and COS cells transfected with GLG-1 and stained with LYPD5-Fcprotein. In one embodiment, the ligand for LYPD5 is the golgi complexlocalized glycoprotein 1 (GLG-1) or E-selectin 1 (ESL-1) polypeptide asshown in SEQ ID NOS:18, 20, 22, or 24, encoded by the nucleic acid shownas SEQ ID NOS:17, 19, 21, or 23, respectively. In another embodiment,the polynucleotide encoding a GLG-1 polypeptide comprises at least 15,at least 25, at least, at least 50, at least 100, at least 250, at least500, at least 750, at least 1000, at least 1250, at least 1500, at least1750, at least 2000, at least 2040, at least 2090, at least 2150, atleast 2200, at least 2300, at least 2400, at least 2500, at least 2600,at least 2700, at least 2800, at least 2900, at least 3000, at least3100, at least 3200, at least 3300, at least 3400, at least 3500, atleast 3600, at least 3700, or at least 3720 contiguous nucleotides ofSEQ ID NOs 17, 19, 21, or 23, or the polynucleotide encoding a GLG-1comprises SEQ ID NOs 17, 19, 21, or 23. In one embodiment, apolynucleotide that binds a polynucleotide encoding a GLG-1 (SEQ IDNOs:17, 19, 21, or 23), or fragment thereof, has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least99% or 100% sequence identity with the GLG-1 polypeptide or fragmentthereof. In one embodiment, the GLG-1 polypeptide comprises at least 10,at least 25, at least 50, at least 75, at least 100, at least 125, atleast 150, at least 175, at least 200, at least 225, at least 250, atleast 275, at least 300, at least 325, at least 350, at least 400, atleast 450, at least 500, at least 550, at least 600, at least 650, atleast 700, at least 750, at least 800, at least 850, at least 900, atleast 950, at least 1000, at least 1050, at least 1100, at least 1150,or at least 1200 contiguous amino acids of SEQ ID NOs: 18, 20, 22, or24, or the GLG-1 polypeptide comprises SEQ ID NOs: 18, 20, 22, or 24.GLG-1 or ESL-1 is expressed on neutrophils, believe to be involved inextravasation of neutrophils into tissues, and thought to play animportant role in inflammation (see Hidalgo et al. (2007) Immunity,26(4): 477-489 incorporated herein by reference in its entirety). GLG-1or ESL-1 has 14 cysteine rich GLG1 domains. The extracellular domain(ECD) is lengthy and as described below, variants or fragments of theGLG-1 ECD were found to have the ability to bind LYPD5.

In another embodiment, the LYPD5 ligand is a variant or fragment a GLG-1or ESL-1 molecule described herein. As shown in FIG. 33A-B, GLG-1 orESL-1 may be viewed as fragments 1, 2, 3, and 4 and as described inExample 11, any one of the 4 fragments are sufficient for LYPD5 binding.

In another embodiment, the LYPD5 ligand is a variant or fragment ofGLG-1 or ESL-1 that is a single GLG-1 domain. As shown in FIG. 34A-B,GLG-1 is made up of multiple GLG-1 domains and as described in Example11, single GLG-1 domains are sufficient for LYPD5 binding.

In another embodiment, the LYPD5 ligand is a variant or fragment ofGLG-1 or ESL-1 that is specific for LYPD5. As shown in FIG. 35A-B, GLG-1includes domains 26-114, domain 115, and domain 150 and as described inExample 11, domain 115 binds LYPD5 but domains 26-114 does not bindLYPD5.

The present invention contemplates variants of GLG-1 in the same mannerit contemplates variants for LY6 family members.

C. Polypeptide Variants

In addition to the polypeptides, antibodies and LY6 binding polypeptidesdescribed herein, it is contemplated that variants of such molecules canbe prepared for use with the invention herein. Such variants can beprepared by introducing appropriate nucleotide changes into the encodingDNA, and/or by synthesis of the desired antibody or polypeptide. Thoseskilled in the art will appreciate that amino acid changes may alterpost-translational processes of these molecules, such as changing thenumber or position of glycosylation sites or altering the membraneanchoring characteristics.

Variations in amino acid sequence can be made, for example, using any ofthe techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the amino acid sequence that results in a change in theamino acid sequence as compared with the native sequence. Optionally thevariation is by substitution of at least one amino acid with any otheramino acid in one or more of the domains of the amino acid sequence ofinterest. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the amino acidsequence of interest with homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. Amino acid substitutions can be the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. The variationallowed may be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

Fragments of the various polypeptides are provided herein. Suchfragments may be truncated at the N-terminus or C-terminus, or may lackinternal residues, for example, when compared with a full length nativeantibody or protein. Such fragments which lack amino acid residues thatare not essential for a desired biological activity are also useful withthe disclosed methods.

The above polypeptide fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating such fragmentsby enzymatic digestion, e.g., by treating the protein with an enzymeknown to cleave proteins at sites defined by particular amino acidresidues, or by digesting the DNA with suitable restriction enzymes andisolating the desired fragment. Yet another suitable technique involvesisolating and amplifying a DNA fragment encoding the desired fragmentfragment by polymerase chain reaction (PCR). Oligonucleotides thatdefine the desired termini of the DNA fragment are employed at the 5′and 3′ primers in the PCR. Preferably, such fragments share at least onebiological and/or immunological activity with the corresponding fulllength molecule.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened in order to identify the desiredvariant.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser, Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp, Gln Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys;Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L)Norleucine; Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Leu Pro (P)Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;Norleucine

Substantial modifications in function or immunological identity of theLY6 polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;-   (2) neutral hydrophilic: Cys, Ser, Thr; Asn; Gln-   (3) acidic: Asp, Glu;-   (4) basic: His, Lys, Arg;-   (5) residues that influence chain orientation: Gly, Pro; and-   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)) or other known techniques can be performedon the cloned DNA to produce the anti-LY6 molecule.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,Science, 244:1081-1085 (1989)). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the LY6 polypeptide also may be substituted, generally with serine,to improve the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to such amolecule to improve its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and target polypeptide. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of LY6polypeptides are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of a nativesequence or an earlier prepared variant.

D. Modifications of Polypeptides

Polypeptides and/or antibodies that have been covalently modified mayalso be suitable for use within the scope of this invention. One type ofcovalent modification includes reacting targeted amino acid residues ofsuch antibodies and polypeptides with an organic derivatizing agent thatis capable of reacting with selected side chains or the N- or C-terminalresidues of such antibodies and polypeptides. Derivatization withbifunctional agents is useful, for instance, for crosslinking thepreceding molecules to a water-insoluble support matrix or surface foruse in purification. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the polypeptides or antibodiescomprises altering the native glycosylation pattern of the antibody orpolypeptide. “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence (either by removing the underlying glycosylation siteor by deleting the glycosylation by chemical and/or enzymatic means),and/or adding one or more glycosylation sites that are not present inthe respective native sequence. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

Glycosylation of antibodies and other polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites may be accomplished by altering theamino acid sequence such that it contains one or more of theabove-described tripeptide sequences (for N-linked glycosylation sites).The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of the originalsuch antibody or polypeptide (for O-linked glycosylation sites). Suchantibody or polypeptide sequence may optionally be altered throughchanges at the DNA level, particularly by mutating the DNA encoding thepreceding amino acid sequences at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties is bychemical or enzymatic coupling of glycosides to the polypeptide. Suchmethods are described in the art, e.g., in WO 87/05330 published 11 Sep.1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Removal of carbohydrate moieties may be accomplished chemically orenzymatically or by mutational substitution of codons encoding for aminoacid residues that serve as targets for glycosylation. Chemicaldeglycosylation techniques are known in the art and described, forinstance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987)and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavageof carbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Another type of covalent modification comprises linking to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. The LY6 polypeptide may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

Modifications forming chimeric molecules results from fusions of onepolypeptide to another, heterologous polypeptide or amino acid sequenceare contemplated for use with the present methods.

In one embodiment, such a chimeric molecule comprises a fusion of apolypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of such antibody orpolypeptide. The presence of such epitope-tagged forms of suchantibodies or polypeptides can be detected using an antibody against thetag polypeptide. Also, provision of the epitope tag enables suchantibodies or polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)); and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody (Paborsky et al., Protein Engineering,3(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide(Hopp et al., BioTechnology, 6:1204-1210 (1988)); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); an α-tubulinepitope peptide (Skinner et al., J. Biol. Chem., 266:15163-15166(1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)).

In an alternative embodiment, the chimeric molecule may comprise afusion of a polypeptide with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a preceding antibody or polypeptide in the place of at least onevariable region within an Ig molecule. In a particularly preferredembodiment, the immunoglobulin fusion includes the hinge, CH₂ and CH₃,or the hinge, CH₁, CH₂ and CH₃ regions of an IgG1 molecule. For theproduction of immunoglobulin fusions see also U.S. Pat. No. 5,428,130issued Jun. 27, 1995.

E. Preparation of Polypeptides

The description below relates primarily to production of polypeptides byculturing cells transformed or transfected with a vector containingnucleic acid such antibodies, polypeptides and oligopeptides. The term“polypeptides” may include antibodies, polypeptides and oligopeptides.It is, of course, contemplated that alternative methods, which are wellknown in the art, may be employed to prepare such antibodies,polypeptides and oligopeptides. For instance, the appropriate amino acidsequence, or portions thereof, may be produced by direct peptidesynthesis using solid-phase techniques [see, e.g., Stewart et al.,Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif.(1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of such antibodies,polypeptides or oligopeptides may be chemically synthesized separatelyand combined using chemical or enzymatic methods to produce the desiredproduct.

1. Isolation of DNA Encoding a Polypeptide

DNA encoding a polypeptide may be obtained from a cDNA library preparedfrom tissue believed to possess such antibody, polypeptide oroligopeptide mRNA and to express it at a detectable level. Accordingly,DNA encoding such polypeptides can be conveniently obtained from a cDNAlibrary prepared from human tissue, a genomic library or by knownsynthetic procedures (e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). Alternatively,PCR methodology may be used. [Sambrook et al., supra; Dieffenbach etal., PCR Primer: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1995)].

Techniques for screening a cDNA library are well known in the art. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for LY6 polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which has thecomplete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvGkan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed in suitablecells (e.g., CHO cells).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorsencoding desired polypeptides. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated polypeptideproduction are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells, such as cell cultures of cotton,corn, potato, soybean, petunia, tomato, and tobacco. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for desired polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the respective LY6polypeptide may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The desired polypeptide may be produced recombinantly not only directly,but also as a fusion polypeptide with a heterologous polypeptide, whichmay be a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the DNA encoding the mature sequence that is inserted into thevector. The signal sequence may be a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the signal sequence may be, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published 4 Apr. 1990), or the signal described in WO 90/13646 published15 Nov. 1990. In mammalian cell expression, mammalian signal sequencesmay be used to direct secretion of the protein, such as signal sequencesfrom secreted polypeptides of the same or related species, as well asviral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up nucleicacid encoding the desire protein, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the nucleic acid sequence encoding the desired amino acidsequence, in order to direct mRNA synthesis. Promoters recognized by avariety of potential host cells are well known. Promoters suitable foruse with prokaryotic hosts include the β-lactamase and lactose promotersystems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promotersystem [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], andhybrid promoters such as the tac promoter [deBoer et al., Proc. Natl.Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding the desired protein sequence.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

DNA Transcription in mammalian host cells is controlled, for example, bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

Transcription of a DNA encoding the desired polypeptide may be increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 bp, that act ona promoter to increase its transcription. Many enhancer sequences arenow known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thecoding sequence of the preceding amino acid sequences, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the respective antibody, polypeptide oroligopeptide described in this section.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of the respective antibody, polypeptide or oligopeptide inrecombinant vertebrate cell culture are described in Gething et al.,Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP117,060; and EP 117,058.

4. Culturing the Host Cells

The host cells used to produce the LY6 polypeptide may be cultured in avariety of media. Commercially available media such as Ham's F10(Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma),and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be usedas culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCINJ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammalConveniently, the antibodies suitable for the present method may beprepared against a native sequence polypeptide or oligopeptide, oragainst exogenous sequence fused to DNA and encoding a specific antibodyepitope of such a polypeptide or oligopeptide.

6. Protein Purification

Polypeptides may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of the preceding can be disruptedby various physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desireable to purify the preceding from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the desired molecules. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular antibody, polypeptide or oligopeptide produced for theclaimed methods.

When using recombinant techniques, the LY6 polypeptide can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If such molecules are produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

Purification can occur using, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being the preferredpurification technique. The suitability of protein A as an affinityligand depends on the species and isotype of any immunoglobulin Fcdomain that is present in the antibody. Protein A can be used to purifyantibodies that are based on human γ1, γ2 or γ4 heavy chains (Lindmarket al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended forall mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the antibody comprises a C_(H)3 domain, theBakerbond ABXJresin (J. T. Baker, Phillipsburg, N.J.) is useful forpurification. Other techniques for protein purification such asfractionation on an ion-exchange column, ethanol precipitation, ReversePhase HPLC, chromatography on silica, chromatography on heparinSEPHAROSEJ chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

H. Pharmaceutical Formulations

Therapeutic formulations (“therapeutic agent”) used in accordance withthe present invention may be prepared for storage by mixing thetherapeutic agent(s) having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington: The Science of Practice of Pharmacy, 20th edition, Gennaro,A. et al., Ed., Philadelphia College of Pharmacy and Science (2000)), inthe form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asacetate, Tris, phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN7, PLURONICS7 or polyethylene glycol(PEG). The antibody preferably comprises the antibody at a concentrationof between 5-200 mg/ml, preferably between 10-100 mg/ml.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to the preceding therapeutic agent(s),it may be desirable to include in the formulation, an additionalantibody, e.g., a second such therapeutic agent, or an antibody to someother target such as a growth factor that affects the growth of theglioma. Alternatively, or additionally, the composition may furthercomprise a chemotherapeutic agent, cytotoxic agent, cytokine, growthinhibitory agent, anti-hormonal agent, and/or cardioprotectant. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy, supra.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT7(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Methods for the Diagnosis and/or Treatment of Inflammatory Bowel Disease

To determine LY6 expression in gastrointestinal tissue or cells of amammal, such as a mammal experiencing IBD, various diagnostic assays areavailable. In one embodiment, LY6 polypeptide overexpression may beanalyzed by RT-PCR, in-situ hybridization, microarray analysis, and/orimmunohistochemistry (IHC). Fresh, frozen and/or parafin embedded tissuesections from a gastrointestinal biopsy (such as from the colon or, morespecifically, the sigmoid colon) from a mammal (such as withoutlimitation a human) may be subjected to the RT-PCR, in situhybridization, microarray analysis and/or IHC assay.

Alternatively, or additionally, FISH assays such as the INFORM7 (sold byVentana, Arizona) or PATHVISION7 (Vysis, Illinois) may be carried out onformalin-fixed, paraffin-embedded tissue to determine the extent (ifany) of LY6 expression and/or upregulation in a tissue sample or biopsy.

LY6 expression may be evaluated using an in vivo diagnostic assay, e.g.,by administering a molecule (such as an antibody, oligopeptide ororganic molecule) which binds the molecule to be detected and is taggedwith a detectable label (e.g., a radioactive isotope or a fluorescentlabel) and externally scanning the patient for localization of thelabel.

Currently, depending on the stage of the IBD, treatment involves one ora combination of the following therapies: surgery to remove affectedbowel tissue, administration of therapeutic agents, including withoutlimitation chemotherapy; dietary changes, and lifestyle management.Therapeutic agents or chemotherapeutic agents useful in the treatment ofIBD are known in the art and representative therapeutic andchemotherapeutic agents are disclosed herein.

In particular, combination therapy with palictaxel and modifiedderivatives (see, e.g., EP0600517) is contemplated. The precedingantibody, polypeptide, oligopeptide or organic molecule will beadministered with a therapeutically effective dose of thechemotherapeutic agent. In another embodiment, such antibody,polypeptide, oligopeptide or organic molecule is administered inconjunction with chemotherapy to enhance the activity and efficacy ofthe chemotherapeutic agent, e.g., paclitaxel. The Physicians=DeskReference (PDR) discloses dosages of these agents that have been used intreatment of various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

Therapeutic agents or chemotherapeutic agents are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intracranial, intracerobrospinal, intra-articular,intrathecal, intravenous, intraarterial, subcutaneous, oral, topical, orinhalation routes.

The present invention provides methods that involve a diagnostic stepand a therapeutic treatment step. In one embodiment, the presentinvention provides methods of detecting inflammatory bowel disease (IBD)in a mammalian subject that include the steps of (1) detecting the levelof expression of a nucleic acid or a gene encoding a LY6 polypeptide (a)in a test sample of tissue or cells obtained from the subject, and (b)in a control sample where a higher level of expression of the LY6nucleic acid or gene in the test sample, as compared to the controlsample, indicates the presence of an IBD in the subject from which thetest sample was obtained; and (2) administering to the subject aneffective amount of an IBD therapeutic agent. In one embodiment, the IBDtherapeutic agent is an antagonist of another IBD-associated molecule.The present invention contemplates various IBD-associated molecules thatare differentially expressed in IBD. In one embodiment, theIBD-associated molecule is a molecule that is differentially expressedin an IBD. In another embodiment, the IBD-associated molecule isover-expressed in an IBD. In yet another embodiment, the over-expressedIBD-associate molecule is an integrin. In one other embodiment, theIBD-associated molecule is integrin, beta 7 (ITGB2) (see WO 2006/026759,which is incorporated herein by reference in its entirety) The term “IBDtherapeutic agent” as used herein refers to an antagonist of anIBD-associated molecule. In one embodiment, the IBD therapeutic agent isan antagonist of an integrin. In another embodiment, the IBD therapeuticagent is an antagonist of ITGB7. In yet another embodiment, the IBDtherapeutic agent is an antagonist of the polypeptide shown as SEQ IDNO: 69 encoded by the nucleic acid sequence shown as SEQ ID NO: 68.

J. Articles of Manufacture and Kits

For diagnostic applications, the article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer indicating a use for detecting and expression of LY6 (such as,without limitation LY6, LYPD1, LYPD3, and/or LYPD5) in agastrointestinal tissue or cell of a mammal In one embodiment, themammal is a human. In one embodiment, the tissue or cell isgastrointestinal tissue or cell. In one embodiment, detecting includesquantitation relative to a contro sample. In an embodiment, thecontainer, label or package insert indicates that the gastrointestinaltissue or cells are from colon of a mammal In an embodiment, thecontainer, label or package insert indicates that increased LY6expression relative to a control sample is indicative of IBD, includingwithout limitation CD and/or UC, in the mammal Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic.Additionally, the article of manufacture may further comprise a secondcontainer comprising a buffer or other reagent (such as detectablelabel) useful for carrying out the detection. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, and dyes.

For isolation and purification of LY6 polypeptide, the kit can containthe LY6-binding reagent coupled to beads (e.g., sepharose beads). Kitscan be provided which contain such molecules for detection andquantitation of LY6 polypeptide in vitro, e.g., in an ELISA or a Westernblot. As with the article of manufacture, the kit comprises a containerand a label or package insert on or associated with the container. Thecontainer holds a composition comprising at least one such LY6 bindingantibody, oligopeptide or organic molecule useable with the invention.Additional containers may be included that contain, e.g., diluents andbuffers, control antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

K. Sense and Anti-Sense LY6-Encoding Nucleic Acids

Molecules that would be expected to bind to nucleic acids encoding anLY6 gene include sense and antisense oligonucleotides, which comprise asingle-stranded nucleic acid sequence (either RNA or DNA) capable ofbinding to target LY6 mRNA or DNA sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of the LY6 DNA or its complement. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein is described in, for example,Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

The sense and/or antisense oligonucleotides hybridizable to a LY6 geneare useful, for example, for detecting the presence of LY6 DNA or mRNAin a tissue or cell sample gastrointestinal tissue or cells of mammalaccording to the invention. The sense and/or antisense compounds used inaccordance with this invention may be conveniently and routinely madethrough the well-known technique of solid phase synthesis. Equipment forsuch synthesis is sold by several vendors including, for example,Applied Biosystems (Foster City, Calif.). Any other means for suchsynthesis known in the art may additionally or alternatively beemployed. It is well known to use similar techniques to prepareoligonucleotides such as the phosphorothioates and alkylatedderivatives. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Patents that teach the preparation of such uptake, distribution and/orabsorption assisting formulations include, but are not limited to, U.S.Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899;5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633;5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756,each of which is herein incorporated by reference.

Sense and antisense oligonucleotides include without limitation primersand probes useful in PCR, RT-PCR, hybridization methods, in-situhybridization, and the like.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense RNA or DNA molecules are generally at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length.

EXAMPLES

The following nonlimiting examples are provided for illustrativepurposes and are not intended to limit the scope of the invention.Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cell lines identified in the following examples, and/orthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Materials and Methods

Reagents, cells and mice: IFNγ, TNFα, and IL1β were obtained fromPeprotech™ (Rocky Hill, N.J.). IFNα was obtained from HycultBiotechnology™ (The Netherlands). For crosslinking experiments, anti-KLHcontrol antibody, anti-LY6A (clone E13-161.7 or D7) were obtained fromPharmingen™ (San Diego, Calif.). Anti-LY6C (clone HK1.4) was obtainedfrom Southern Biotech™ (Birmingham, Ala.).

Chronic CD45RB^(high) transfer colitis was induced as describedpreviously in SCID mice on a Balb/c background (Powrie, F. et al.,(1994) Immunity 1:553-562). IL10−/− mice (Kuhn, R. et al., (1993) Cell75:263-274) on a 129 background, which develop spontaneous colitis, weresacrificed between 11 and 13 weeks of age. Colons were snap frozen inOCT until used in experiments as described. Proximal colon, middlecolon, distal colon and rectum were scored using a scale of 0-5(0=normal bowel, 5=severe disease). Scores were summed to achieve atotal colitis severity score for each animal.

The young adult mouse colonocyte (YAMC) cell line (provided by RobertWhitehead, Vanderbilt University Medical Center, Nahville, Tenn.) wasderived from the Immortomouse™ a transgenic animal containing atemperature-sensitive T-antigen (tsTag) under the control of aninterferon-γ-dependent promoter, as previously described (Whitehead, R.H. et al, (1993) Proc Natl Acad Sci USA 90:587-591). YAMC cellsproliferate under permissive conditions of 32° C. in the presence of 5units/ml IFN-γ (Peprotech™, New Jersey), but no longer proliferate uponremoval of IFN-γ at 37° C. (nonpermissive conditions).

YAMC cells were cultured in RPMI containing 5% FBS, 2 mM L-glutamine,penicillin/streptomycin, 5 U/ml IFNγ and N-2 supplement (Invitrogen™,Carlsbad, Calif.). Cells were cultured under non-permissive conditionsfor 24 hours prior to experiments, and for the duration ofexperimentation.

CMT93 cells were obtained from ATCC (ATCC Number® CCL-223™, ATCC,Manassas, Va.) cultured in DMEM containing 10% FBS, 2 mM L-glutamine,and penicillin/streptomycin.

Laser capture microscopy and RNA purification: 10-12 μm sections wereapplied to LCM membrane slides (Molecular Machines™, Glattbrugg,Switzerland). Slides were subjected to an abbreviated H&E stain (totaltime of about five minutes) before crypt epithelial cells werehistologically identified and dissected using an MMI Cellcut™ microscope(Molecular Machines, Glattbrugg, Switzerland). RNA was purified from thedissected cells using the Arcturus™ Picopure™ RNA purification kit andmanufacturer's protocols (Arcturus™, Sunnyvale, Calif.) and quantifiedusing the NanoDrop ND-1000™ Spectrophotometer (NanoDrop Technologies™,Wilmington, Del.).

Microarray hybridization and data analysis: The quantity and quality ofinput total RNA samples was determined using ND-1000 spectrophotometer(NanoDrop™ Technologies, Montchanin, Del.) and Bioanalyzer 2100™(Agilent™ Technologies, Palo Alto, Calif.), respectively. The method forpreparation of Cy-dye labeled cRNA and array hybridization was providedby Agilent™ Technologies (Palo Alto, Calif.). Briefly, total RNA samplewas converted to double-stranded cDNA and then to labeled cRNA using aLow RNA Input Fluorescent Linear Amplification™ Kit (Agilent™, Product#5184-3523). The labeled cRNA was purified using RNeasy™ mini kit(Qiagen™, San Diego, Calif.) and then quantified using ND-1000™spectrophotometer (Nanodrop™, Technologies). Cy-dye incorporation wasdetermined by running the labeled cRNA on a Novex™ TBE-Urea gel(Invitrogen™, Carlsbad, Calif.) followed by gel scanning on a Typhoon™0scanner (GE Healthcare™, Piscataway, N.J.). To determine the amount ofCy-dye fluorescent counts, the gel images were analyzed usingImageQuant™ software (GE Healthcare™). Approximately 500,000 counts ofCy-dye labeled cRNA was fragmented and hybridized to the Agilent's wholemouse genome array as described in Agilent's In situ Hybridizationkit-plus (Agilent™, Product #5184-3568). LCM samples were labeled withCy5 dye and hybridized against Cy3 dye labeled universal mouse reference(Stratagene™, La Jolla, Calif.). Following hybridization, the arrayswere washed, dried with acetonitrile and scanned on the Agilent™ DNAmicroarray scanner. The array image files were analyzed using Agilent™'sFeature Extraction™ software 7.5 and further data analysis was performedusing Resolver™ (Merck™, Seattle, Wash.).

Data was analyzed using Rosetta Resolver™ software (RosettaBiosoftware™, Seattle, Wash.). Briefly, healthy and colitic samples weregrouped separately and probes that passed two-tailed anova (p<0.05) wereselected. These probes were analyzed further for probes thatdemonstrated a two fold or greater change in colitic samples versushealthy samples.

Real time quantitative RT-PCR: RT-PCR was performed on extracted RNAusing Taqman™ Gold™ RT-PCR kit and reagents (Applied Biosystems™, FosterCity, Calif.). All samples were run with gene specific primers using5′-FAM and 3′-TAMRA labeled internal probes. Analysis was performedcompared to housekeeping gene, SPF31, specific primers by the 2^(−ΔΔ)Ctmethod as described (Livak, K. J., and T. D. Schmittgen (2001) Methods25:402-408). Primers and probes were either designed using Primer3™software (Rozen, S., and H. Skaletsky (2000) Methods Mol Biol132:365-386) or obtained commercially (Applied Biosystems™). Primers andprobes used for these assays were the following, shown in the 5′-3′direction:

LY6A: (SEQ ID NO: 39) Sense: CTT ACC CAT CTG CCC TCC TA (SEQ ID NO: 40)Antisense: CCT CCA TTG GGA ACT GCT AC (SEQ ID NO: 41)Probe: TCC TGT TGC CAG GAA GAC CTC TGC LY6C: (SEQ ID NO: 42)Sense: ACT TCC TGC CCA GCA GTT AC (SEQ ID NO: 43)Antisense: GGC ACT GAC GGG TCT TTA GT (SEQ ID NO: 44)Probe: CTG CCG CGC CTC TGA TGG AT

Immunofluorescent staining: Frozen tissues were cut into 5 μm sectionsand stained with biotinylated anti-LY6C (Southern Biotech™, Birmingham,Ala.) or anti-SCA-1 at 2.5 ng/ml (R&D Systems™, Minneapolis, Minn.).Slides were washed and labeled with Alexa Fluor™ 488 conjugatedstreptavidin, mounted using Prolong Gold™ with DAPI (Invitrogen™,Carlsbad, Calif.) and visualized by confocal microscopy.

Crosslinking LY6 molecules: The ability of crosslinked LY6 polypeptideto effect chemokine production was tested by incubating YAMC cells withplate-bound anti-LY6C or anti-KLH (control) antibodies and measuring theproduction of chemokines CXCL2, CXCL5, and CCL7. Because lipid raftformation in the cell membrane is required for crosslinking, chemokineproduction was tested under conditions of normal raft formation(non-cholesterol depletion) and under conditions of cholesteroldepletion.

For crosslinking using plate-bound antibody, 100 μl of anti-LY6C oranti-KLH (control) antibody at 5 μg/mL concentration was added to a 96well plate, or 2 mL were added to a 60 mm² dish and allowed to bind tothe plate for 15 hours at 4° C. YAMC cells, grown in cholesteroldepleting or non-depleting conditions (as provided in Example 5, herein)were incubated with the plate-bound antibodies for 15 hours at 32° C.under cholesterol non-depleting conditions RNA was collected andexpression levels of CXCL2, CXCL5, and CCL7 were determined. The assayis further described and results are shown in Example 5, herein.

siRNA inhibition: Individual siRNA directed against murine LY6C wereobtained from Dharmacon (Lafayette, Colo.). SiRNA was transfected intoYAMC cells using lipofectamine 2000 (Invitrogen) and standard protocols.72 hours after transfection, cells were collected to determine knockdownefficiency. One siRNA was chosen for crosslinking experiments based onsuperior knockdown efficiency (95% inhibition by quantitative RT-PCR).

CXCL5 secretion: Supernatants were collected at the indicated time pointfrom stimulated cells and cytokine CXCL5 concentrations were determinedby ELISA using a commercially available kit from R&D Systems™ andmanufacturer's protocols. The level of detection was 15 pg/ml of CXCL5.

Cholesterol depletion: YAMC cells were cultured for 72 hours in serumfree medium at 37° C. in the presence of 4 μM lovastatin and 250 μMmevalonate (Sigma). Cells were plated and maintained in lovastatin andmevalonate throughout the experiment.

Statistics: Student's t test was used for comparison between groups (*indicates p<0.05).

Example 2 Gene Expression Patterns of IEC are Altered During Colitis

Studies have indicated that gene expression patterns of IEC aresignificantly altered in mouse models of colitis, as well as human IBD(Fahlgren, A., et al. (2004) Clin Exp Immunol 137:379-385; Brand, S. etal. (2006) Am J Physiol Gastrointest Liver Physiol 290:G827-838; Ruiz,P. A. et al. (2005) J Immunol 174:2990-2999). In this example, evaluatedgene expression patterns in IEC of healthy and colitic mice wereexamined in order to illuminate novel genes and pathways altered in IBD.

The identification of genes involved in the immunopathology of IBD wassought by evaluating intestinal epithelial cells (IEC) from theCD45RB^(Hi) T cell transfer colitis mouse model as well as theIL10^(−/−) mouse model, both of which result from Th1 dysregulation andshare many features of human Crohn's disease (Elson, C. O. et al. (2005)Immunol Rev 206:260-276; Bouma, G., and W. Strober (2003) Nat RevImmunol 3:521-533). Laser capture microdissection (LCM) was used toisolate crypt IEC from the colons of healthy and colitic mice in the twomodels of murine IBD. RNA was extracted from these samples and analyzedby microarray technology as described herein in Example 1. The geneexpression profile of IEC of colitic mice in the transfer colitis modelidentified 1770 probes with >2 fold expression changes compared tocontrol mice, while the IL10−/− model identified 1140 probes.Overlapping in both models, there were 540 probes with >2 fold changesin expression, corresponding to approximately 400 different genes (datanot shown).

Example 3 Pathways and Genes Affected in IEC During Colitis

Of the approximately 400 genes affected in both models, genes involvedin antigen presentation, TLR signaling and cell migration wereoverrepresented (Table 7). In Table 7, numbers represent the mean withstandard deviation of the fold change compared to universal standard RNAof colitic mice over healthy mice in either the IL10^(−/−) model ofcolitis or the CD45RB^(Hi) model of colitis, as indicated. The resultsindicated that some IEC expressed genes show altered expression patternsin murine models of IBD. Many of these genes, including TLR2, CCL7,CXCL5 and ICAM-1 have been described previously as having increasedepithelial expression during colitis (Breider, M. A. et al. (1997) VetPathol 34:598-604; Uguccioni, M. et al. (1999) Am J Pathol 155:331-336;Z'Graggen, K. et al. (1997) Gastroenterology 113:808-816; Singh, J. C.et al. (2005) Am J Physiol Gastrointest Liver Physiol 288:G514-524),suggesting that the gene expression pattern obtained in thesemicroarrays are an accurate reflection of the biology of IEC in colitis.

TABLE 7 Fold change (p value): IL10 −/− model CD45RBhi model Cellmigration CXCL1 +3.89 (<0.0001) +2.09 (0.00066) CXCL5 +21.82 (<0.0001) +23.34 (<0.0001) CXCL13 +3.01 (<0.0001) +2.85 (<0.0001) CCL6 −3.47(<0.0001) −2.5 (<0.0001) CCL7  +4.2 (<0.0001) +5.54 (0.00026) CCL11−3.43 (<0.0001) −3.6 (0.00607) TLR signaling TLR2 +2.15 (<0.0001) +2.68(<0.0001) Fos +3.64 (<0.0001) +2.03 (<0.0001) LBP +2.34 (<0.0001) +2.57(<0.0001) NFKBIA +2.37 (<0.0001) +2.15 (<0.0001) Antigen presentationH2-D1 +2.77 (<0.0001) +2.23 (<0.0001) HLA-A +2.83 (<0.0001) +2.40(<0.0001) HLA-B +2.71 (<0.0001) +2.44 (<0.0001) HLA-E +2.31 (<0.0001)+2.34 (<0.0001) ICAM-1 +2.51 (<0.0001) +2.587 (<0.0001) PSMB8 +8.10(<0.0001) +3.09 (<0.0001) PSMB9 +6.61 (<0.0001) +2.72 (<0.0001) TAP1+4.05 (<0.0001) +4.10 (<0.0001) TAP2 +2.08 (<0.0001) +2.18 (<0.0001)

IEC can function as non-professional APC (Snoeck, V. et al., (2005)Microbes Infect 7:997-1004; and Shao, L et al., (2005) Immunol Rev206:160-176), and the gene expression pattern obtained in thesemicroarrays indicate that these functions are enhanced during colitis byupregulation in genes associated with antigen processing, such as LMP7and TAP1, as well as MHC class I and II genes which would serve toenhance presentation of antigens on the surface of the IEC.

The microarray data supports the concept that colitic IEC attract immunecells to the colon through altered chemokine expression, and may presentantigen to infiltrating T cells by upregulating expression of genesassociated with antigen presentation.

Example 4 Expression of LY6 Family Members is Strongly Upregulated onthe Surface of Colitic IEC

Members of the mouse LY6 family of molecules were overrepresented innumber as well as degree of upregulation in both the transfer colitismouse model and the IL10−/− mouse model (FIGS. 23A and 23B). Theseresults were confirmed by real-time quantitative RT-PCR of pooled andamplified IEC RNA in the transfer colitis model (data not shown).Expression of the LY6 family members was unique to the disease state, sono healthy mice expressed appreciable levels of any of these LY6 familymembers.

While expression of murine LY6 molecules on the surface of cells ofhematopoietic origin is known, expression on IEC has not been previouslydescribed (Bamezai, A. (2004) Arch Immunol Ther Exp (Warsz) 52:255-266;and Rock, K. L. et al. (1989) Immunol Rev 111:195-224). Expression ofmurine LY6A and LY6C is detectable on many non-epithelial cells presentwithin the colon, such as T cells and granulocytes. Immunofluorescentstaining was performed for both murine LY6A and LY6C on healthy andcolitic colons. Levels of murine LY6A and LY6C were minimal or absent onthe surface of healthy IEC (FIGS. 24A and 24C, respectively). Expressionof both murine LY6A and LY6C was detectable on the surface of IECthroughout the colons of colitic mice (FIGS. 24B and 24D, respectively).There was no evidence of polarization of either LY6A or LY6C, andstaining was present on both the apical and basolateral membranes,making LY6 molecules potentially accessible to ligands on eithersurface. These results indicate that the microarray analysis resultsshowing upregulation of murine LY6A and LY6C in murine colitic modelswas not due to the influx of contaminating immune cells.

Example 4 Transcription of LY6 Genes is Stimulated by InflammatoryCytokines

LY6 expression on T cells is induced and enhanced by both type I andtype II IFNs (Khodadoust, M. M., K. D. Khan, and A. L. Bothwell. 1999.Complex regulation of Ly-6E gene transcription in T cells by IFNs. JImmunol 163:811-819). Furthermore, expression of a number of cytokines,is elevated in the colon during active colitis (Niessner, M., and B. A.Volk. 1995. Altered Th1/Th2 cytokine profiles in the intestinal mucosaof patients with inflammatory bowel disease as assessed by quantitativereversed transcribed polymerase chain reaction (RT-PCR). Clin ExpImmunol 101:428-435).

To determine if cytokines present during colitis affect transcription ofLY6 family members in IEC, we treated YAMC cells, a conditionallyimmortalized murine IEC line, with IL-1β, IFNα, TNFα, IFNγ or thecombination of TNFα and IFNγ and analyzed the transcription of allidentified murine LY6 genes by real-time quantitative RT-PCR (Table 8).Briefly, mRNA levels of the indicated LY6 family member in IEC wasdetermined by real time quantitative RT-PCR after 15 hours of treatmentwith the indicated cytokine. Number represents the fold change(determined by 2^(−ΔΔCt) method) versus the untreated, media control. *,P<0.05 versus media control. †, p<0.05 versus IFNγ treated cells. Thefollowing LY6 family members were tested, but not detected in samples,regardless of treatment: LY6K, Lypd3, Lypd4, Lypd5, LY6g5b, Ly6g6d,Ly6g6e, Slurp1. The results indicate that IEC upregulate LY6 familymembers in response to inflammatory cytokines.

TABLE 8 Media IL1β TNFα IFNα IFNγ IFNγ & TNFα Ly6A 1.0 1.8* 2.2* 2.8*33.1* 65.4*† Ly6C 1.0 1.6* 1.2* 2.4* 65.6* 63.6* Ly6D 1.0 2.7* 2.1* 1.5*1.0 0.9 Ly6E 1.0 1.4* 1.5* 2.1* 1.9* 2.9*† Ly6F 1.0 2.5* 0.6* 7.1*108.2* 169.7*† Ly6H 1.0 1.0 1.1 1.2 3.7* 1.4† Lypd1 1.0 1.5* 2.1* 1.3*1.3* 2.9*† Lypd2 1.0 0.1* ND 0.4* 0.1* ND Ly6g5c 1.1 1.3 0.8 0.9 1.3 1.1Ly6g6c 1.0 0.7 0.7 0.6 0.6* 0.3*† Slurp2/ 1.1 0.7 0.4 0.7 1.5 0.4* Lynx1

While many of the LY6 family members were not detected in either thepresence or absence of inflammatory cytokines, we detected a strongupregulation in the transcription of murine LY6A, LY6C and LY6F inresponse to the majority of the cytokines tested, as well as moremoderate upregulation of murine LY6E, LY6H and LYPD1 in response to somecytokines tested. However, IFNγ was by far the most potent cytokine ininducing LY6 upregulation. Furthermore, TNFα enhanced the effects ofIFNγ on the expression of LY6A, LY6F, LY6E and LYPD1. Similarupregulation of LY6 family members were seen in another murine IEC line,CMT93 (data not shown).

To examine the surface expression of LY6 family members in response tocytokines, YAMC cells were exposed to the above cytokines and analyzedby flow cytometry for expression of murine LY6A and LY6C, for whichcommercial antibodies are available, as described herein in Example 1.High levels of murine LY6A was expressed on YAMC cells even in theabsence of added cytokines (FIG. 25B, media). Expression of murine LY6C(FIG. 25A, media) was considerably lower than expression of LY6A.

IL-1β and TNFα induced slight increases in the surface expression ofboth murine LY6A and LY6C, in agreement with the RNA expression (FIGS.25A and 25B). A more moderate increase in expression was noted when IFNαwas added to the cells, while IFNγ induced dramatic increases in surfaceexpression of both LY6A and LY6C (FIGS. 25A and 25B). Surface proteinexpression closely mirrored RNA expression. Th2 cytokines, such as IL4,IL10 or IL13 had no effect on surface expression of either LY6A or LY6C(data not shown).

Induction of both LY6A (FIG. 25D) and LY6C (FIG. 25C) by IFNγ was dosedependent. Doses as low as 6.25 units/ml of IFNγ resulted in detectableincreases in both LY6 molecules by flow cytometry. Furthermore, theincrease in both LY6A (FIG. 25F) and LY6C (FIG. 25E) surface expressionbecame evident between 2 and 4 hours after IFNγ treatment, and steadilyincreased for at least 24 hours after IFNγ treatment. This dataindicates that relatively low concentrations of IFNγ are sufficient toincrease surface expression of LY6 molecules within hours.

There is evidence that IL-22, which is secreted primarily from activatedT cells, functions through the IL-22R complex, present on IEC to promotecytokine production and an inflammatory phenotype (Brand, S. F. et al.Am J Physiol Gastrointest Liver Physiol 290:G827-838 (2006)).Furthermore, IL-22 is involved in the immunopathogenesis of Crohn'sDisease. To examine whether IL-22 affects LY6 molecule expression onmurine IEC, YAMC cells were cultured in the presence of IL-22 andanalyzed for expression of LY6C (FIG. 25G) and LY6A ((FIG. 25H). BothLY6 molecules were substantially increased in the presence of IL-22 atcomparable levels to the induction seen after treatment with IFNγ.

To ensure that the upregulation of LY6 molecules was not specific to theYAMC cell line, RNA levels of murine LY6A and LY6C in the murine colonicepithelial tumor cell line CMT93 was examined. Levels of both murineLY6A and murine LY6C were upregulated upon treatment with IFNγ (FIG.25I). Though the levels of upregulation of LY6 molecules were moremodest in CMT93 cells, flow cytometry analysis indicated that levelswere quite high even in untreated cells (data not shown), which islikely a result of the tumor phenotype of CMT93 cells.

This data supports the data obtained by real time quantitative RT-PCR inconfirming that IEC upregulate LY6 family members in response toinflammatory cytokines.

Example 5 LY6 Stimulation of IEC is Associated with Lipid Raft Formation

As GPI-anchored proteins, LY6 family members do not possess a uniqueintracellular domain associated with traditional outside-in signaling.Rather, they are present within lipid raft microdomains (Bohuslav, J. etal. Eur J Immunol 23:825-831(1993)). However, it has been suggested thatcross-linking of LY6 family members on the surface of cells results inredistribution of other cell surface molecules as well as reorganizationof lipid raft structures, suggesting a mechanism by which LY6 moleculescan affect signal transduction and downstream cellular functions(Simons, K. et al., Nat Rev Mol Cell Biol 1:31-39 (2000)).

Few ligands for LY6 proteins have been identified to date, and no ligandfor LY6A or LY6C is currently known (Paret, C. et al., (2005) Int JCancer 115:724-733; Apostolopoulos, J. et al., (2000) Immunity12:223-232; and Classon, B. J. (2001) Trends Immunol. 22:126-127).Cholesterol is required to maintain lipid raft integrity. (Simons, K.,et al. J Clin Invest 110:597-603 (2002)), and depletion of cholesterolis often used to inhibit lipid raft biosynthesis in vitro (von Tresckow,B. et al. J Immunol 172:4324-4331 (2004)).

To analyze whether lipid raft reorganization occurs in IEC in responseto LY6 crosslinking, YAMC cells were grown in cholesterol-depletingconditions (conditions under which lipid rafts are depleted from cells)and cholesterol non-depleting conditions (conditions permissive forlipid raft formation). For cholesterol depleting conditions, YAMC cellswere grown in the absence of serum and in the presence of 4 μMlovastatin and 0.25 mM mevalonate (Sigma Chemical Co., St. Louis, Mo.)for 72 hours at 37° C. The same growth conditions were use for YAMCcells under cholesterol non-depleting conditions, except that nolovastatin or mevalonate were added to the growth medium. Cells werethen lifted and LY6C was crosslinked as described above in Example 1.RNA was collected and expression levels of CXCL2, CXCL5, and CCL7 weredetermined.

The results of these studies indicated that lipid raft depletion resultsin an inhibition of LY6C-mediated chemokine production. FIGS. 26A-26Cshow that cholesterol depleted (dark bars) YAMC cells produced lesschemokine than cells that were not depleted of cholesterol (open bars).Cholesterol depletion affected chemokine production in control anti-KLHstimulated groups, irrespective of LY6C stimulation, however theresponse was minimal and not in a consistent direction. To examinewhether cholesterol depletion globally affected cell viability, wemeasured cell death, by 7AAD exclusion, and determined that cholesteroldepletion did not significantly affect the viability of the YAMC cells(92% viability versus 86% in the cholesterol depleted cells, data notshown). Surface expression of both LY6A (FIG. 26D) and LY6C (FIG. 26E)were both significantly lower in cholesterol depleted YAMC cells,suggesting that plasma membrane cholesterol levels and lipid raftintegrity affect the levels of LY6 expression on the surface of cells.This data suggests that lipid raft integrity, influenced by cholesterolbiosynthesis, allows for the expression of LY6 molecules on the surface,and is potentially involved in the LY6C mediated induction ofchemokines. Thus, the enhancement of chemokine production mediated byinteraction of LY6C polypeptides in the cell membrane requires thepresence of lipid rafts on the cell surface.

Example 6 Crosslinking LY6C Results in Increased Surface Expression ofLY6 Molecules

It has been reported that crosslinking LY6C on the surface of T cellsresults in shedding of LY6C (Jaakkola, I. et al. (2003) J Immunol170:1283-1290). However, unlike T cells, when murine LY6C wascrosslinked on the surface of IEC, no shedding of either LY6A or LY6Coccurred (FIGS. 27A and 27B, respectively). To the contrary, in theabsence of IFNγ, surface expression levels of both LY6A and LY6C wereincreased on IEC with crosslinked LY6C, but not LY6A. When IEC werepreincubated with IFNγ, much of this effect was abolished (FIG. 27C),however a slight upregulation of LY6A was still detected (FIG. 27D).

These data indicate a positive feedback loop whereby stimulation throughLY6C on IEC results in increased surface expression of LY6 molecules.

Example 7 Stimulation of LY6A Results in Increased Secretion ofChemokines

Functions for LY6 molecules have not been fully elucidated. To examinethe role of LY6 molecules in the immunopathology of colitis, stimulationof LY6 molecules was studied for affects on the transcription andsecretion of chemokines from IEC.

To analyze production of chemokines from IEC in response to crosslinkingof murine LY6 molecules, YAMC cells, either pretreated with IFNγ oruntreated, were cultured on plates coated with either anti-KLH controlantibody, anti-LY6A or anti-LY6C. Twenty four hours later, mRNA fromthese cells was obtained and analyzed by quantitative RT-PCR forexpression of CCL2, CCL4, CCL5, CCL7, CCL8, CCL25, CXCL1, CXCL2, CXCL5,CXCL10, CXCL12 and CX3CL1, which are chemokines that have beenimplicated in colitis (Table 9) (Papadakis, K. A. (2004) Curr AllergyAsthma Rep 4:83-89; Banks, C. et al., (2003) J Pathol 199:28-35; andPapadakis, K. A., and S. R. Targan (2000) Inflamm Bowel Dis 6:303-313).The assay was performed under non-permissive growth conditions (37° C.in the absence of IFNγ) to rule out the possibility of increasedproliferation of IEC in response to IFNγ stimulation.

TABLE 9 Pre-treatment Media IFN Crosslink Anti- Anti- Anti- Anti- Anti-Anti- KLH LY6A LY6C KLH LY6A LY6C CCL2 1.01 −1.38 8.81 3.53 2.83 16.12(0.14) (0.13) (0.72) (0.21) (0.21) (0.56) CCL4 1.31 1.83 3.15 5.35 3.6516.12 (1.14) (0.49) (1.17) (0.48) (0.69) (0.56) CCL5 1.00 1.09 3.31 2.732.76 10.13 (0.10) (0.02) (0.15) (0.13) (0.23) (0.27) CCL7 1.00 1.06 3.372.82 1.39 5.81 (0.11) (0.08) (0.15) (0.44) (0.33) (0.51) CCL8 1.03 2.0512.78 74.22 74.44 110.44 (0.30) (0.37) (3.14) (8.94) (9.81) (3.36) CCL251.01 1.06 1.16 1.38 1.32 1.46 (0.16) (0.11) (0.00) (0.36) (0.15) (0.19)CXCL1 1.00 −3.17 11.58 −1.13 −1.64 13.36 (0.07) (0.11) (0.12) (0.10)(0.17) (0.35) CXCL2 1.33 ND 21.81 14.30 10.95 113.20 (1.10) (3.13)(3.30) (3.05) (16.23) CXCL5 1.08 ND 118.45 1.70 ND 150.99 (0.53) (65.14)(1.15) (55.50) CXCL10 1.00 1.02 5.11 5.68 5.22 12.22 (0.05) (0.06)(0.19) (0.31) (0.22) (0.51) CXCL12 1.01 1.12 −1.99 −1.11 −1.23 −3.02(0.14) (0.05) (0.14) (0.05) (0.21) (0.06) CX3CL1 1.00 −1.18 1.92 2.211.87 3.22 (0.08) (0.15) (0.07) (0.11) (0.16) (0.42)

Cells pretreated with IFNγ showed upregulation of many of thesechemokine genes (see Media, Anti-KLH group versus IFNγ, Anti-KLH groupof Table 9). However, with the exception of an upregulation of CCL8 anda downregulation of CXCL1, anti-LY6A stimulated YAMC cells showedsimilar gene expression patterns as anti-KLH stimulated YAMC cells.However, YAMC cells stimulated with anti-LY6C showed increasedexpression of all chemokines analyzed except for CCL25, which remainedessentially unchanged, and CXCL12, which was downregulated in responseto LY6C stimulation. While the increased gene expression of chemokinesinduced by LY6C crosslinking was not dependent upon IFNγ, cellspretreated with IFNγ showed increased expression of chemokines versuscells that had not been pretreated with IFNγ.

To analyze the kinetics of chemokine induction induced by murine LY6Cstimulation, 96 well plates were coated with anti-KLH antibody or eitheranti-LY6A or anti-LY6C monoclonal antibodies. YAMC cells, eitherpretreated or not with IFNγ, were added for 24, 48 or 72 hours. At theindicated time point RNA was collected for quantitative RT-PCR analysisand supernatants were collected for ELISA.

Within 24 hours, a spike in transcription of both CXCL5 and CCL7 wasdetected on cells with crosslinked LY6C, but not LY6A (FIG. 28A).Increased expression of CXCL5 and CCL7 diminished over time but wasstill detectable after 72 hours in culture. Though IFNγ was not requiredto enhance chemokine transcription, IFNγ acted synergistically with LY6Cstimulation in inducing transcription of both CXCL5 and CCL7 at earlytime points.

In parallel with the gene expression, supernatants of LY6C, but notLY6A, crosslinked cells contained significantly higher concentrations ofCXCL5 at 48 hours (FIG. 28B). The effect was dose dependent, anddetectable with as little as 1 μg/ml of coated anti-LY6C. Liketranscription, secretion of CXCL5 was enhanced when cells werepretreated with IFNγ, but IFNγ was not required for the effect.Increased secretion of CXCL5 was noted at both the 24 and 72 hour timepoints as well.

To ensure that LY6C was involved in the observed upregulation ofchemokines, we used siRNA to knockdown LY6C. LY6C transcript wasinhibited by 95% in the absence of IFNγ and about 90% in the presence ofIFNγ by real time quantitative RT-PCR which corresponded tosignificantly lower levels of LY6C on the surface of the YAMC cells(data not shown). Cells with decreased levels of LY6C on the surfaceshowed a diminished response to LY6C crosslinking with regard totranscription of chemokines (FIG. 28C). Secretion of CXCL5 was markedlyinhibited by knocking down LY6C as well (data not shown).

These results indicate that crosslinking of LY6C, but not LY6A, on thesurface of IEC results in increased secretion of chemokines.

Example 8 IEC in vivo Show a Similar Chemokine Gene Expression to LY6CStimulated Cells

The above data establishes a model whereby IEC stimulated through murineLY6C significantly upregulate expression of chemokine genes.

Analyzing the microarray data from laser capture microdissected IEC inmurine models of colitis, the expression of the same 12 chemokine genesin healthy and colitic mice in the two murine models of colitis wasexamined to determine if the chemokines stimulated by LY6C crosslinkingin vitro correlate with the chemokines secreted by IEC in vivo (FIGS.29A and 29B). Though the expression pattern is not identical to theupregulation of chemokines resulting from LY6C stimulation, expressionof CXCL5, which was the most highly upregulated chemokine gene in invitro studies, was also the highest upregulated chemokine in murinemodels of colitis. We saw significant upregulation in expression ofCXCL1, CXCL10, CCL5 and CCL7 in both models of colitis. In addition, wesaw upregulation of CCL4 and CCL8 in the transfer colitis model or theIL10−/− model, respectively.

Interestingly, the only chemokine that was down-regulated as a result ofmurine LY6C stimulation in vitro, CXCL12 was also the only one of thesechemokines downregulated in vivo.

Example 9 Expression of Human LY6 Genes in Colon Cells

Expression of human LY6H, LYPD1, LYPD3, and LYPD5 in a human colon cellline, Colo 205 cells (a cell line derived from human colon carcinoma,ATCC™ accession number CCL-222™), was examined. Human Colo 205 cellswere treated with the cytokines IFN-r, LPS, TNFα, IFN-r+TNFα, IFN-r+LPS,or LPS+TNFα (all at 100 ng/ml, except LPS at 1 ug/ml) for 18 hours(LYPD3) or 24 hours (LY6H or LYPD5). RNA was collect and purified andexpression of the indicated LY6 family member was determined byquantitative RT-PCR using reagents from Applied Biosystems™ according tomanufacturer's instructions. Primers and probes used for RT-PCR analysiswere the following:

LYPD1: (SEQ ID NO: 59) Sense: CAT GAT CCT CCG AAT CTG GT (SEQ ID NO: 60)Antisense: AGC ACA GAA CAG AGG GGC TA (SEQ ID NO: 61)Probe: ATA CGG CCA ATG TCA CAA CA LYPD3: (SEQ ID NO: 62)Sense: ACT TCC TGT TCC CAC CAC TG (SEQ ID NO: 63)Antisense: AGA GGA CAA GCG GAG AGA CA (SEQ ID NO: 64)Probe: TTC TGG CAG GGG TGT TCT AG LY6H: (SEQ ID NO: 65)Sense: AGC AGC AGC AGG AAG GAT (SEQ ID NO: 66)Antisense: AAA AGT GCC GCT TAA CGA AG (SEQ ID NO: 67)Probe: CAA GAT GTG TGC TTC CTC CTG CGA

LYPD5 primers and probes were purchased from Applied Biosystems™(catalog number HS00289062_m1).

The results plotted in FIGS. 30A-30C indicate fold increases inexpression of these human LY6 genes relative to human B-actin control.Significant increases in expression of human LY6H, LYPD3, and LYPD5 wereobserved following treatment with the indicated cytokines.

Example 10 Expression of Human LY6 Genes in Colon Biopsy Tissue

To further investigate the source of the increased LYPD1 and LYPD5expression in the colon of patients with CD and UC, was undertaken in acohort of biopsies of patients with UC, CD and controls. Microarrayanalysis for LYPD1 expression using RNA extracted from colon biopsiesshowed statistically increased expression in inflamed colon tissue of CDpatients (FIG. 31A). In the UC and CD biopsies taken from the colon,statistically increased LYPD5 expression was observed in inflamed UC andCD patients (FIG. 31B). This was not observed in the non-inflamedcontrol biopsies.

Expression of human LY6H in terminal ileum biopsies of inflamed IBDtissue was analyzed relative to control (non-IBD) terminal ileumbiopsies using RT-PCR (Taqman™) analysis. Human LY6H expression was atleast 1.5 fold greater in inflamed IBD biopsies relative to control.

Human LYPD3 expression in inflamed UC colon biopsies was upregulated andless than 2 fold greater in inflamed IBD biopsies relative to control.

The results of these examples demonstrated expression of LY6 moleculeson the surface of IEC, and further indicated that expression is uniqueto IEC in the context of inflammation. Furthermore, surface expressionlevels of LY6A and LY6C were high on IEC of colitic mice, and nearlyuniversal throughout the colon. As molecules both specific to thediseased state, and ubiquitously expressed during disease, detection ofhuman LY6 gene or polypeptide expression, particularly human LY6H,LYPD1, LYPD3, and LYPD5, is a useful method for detecting IBD, includingUC and/or CD in humans. Additionally, the method of detecting human LY6expression is useful for diagnosing IBD, UC and/or CD in a human andmonitoring response to IBD therapeutic agents.

In the Examples disclosed herein, the functional significance of LY6expression in IEC was demonstrated. YAMC cells were strongly positivefor LY6A, and expressed lower levels of LY6C. However, upon stimulationwith a number of cytokines present within the colon during colitis,including IL-1β, TNFα, IFNα, and in particular IL-22 and IFNγ,expression levels of both LY6 molecules were greatly enhanced. YAMCcells pretreated with IFNγ to upregulate expression of LY6 molecules,were a useful in vitro model to analyze functional significance for LY6expression.

The conditionally immortalized nature of the YAMC cells comes from MHCII promoter driven expression of the SV40 large T antigen; low levels(2.5-5 U/ml) of IFNγ are used to drive proliferation of these cells(Whitehead, R. H. et al. (1993) Proc Natl Acad Sci USA 90:587-591;Whitehead, R. H., and J. L. Joseph. (1994) Epithelial Cell Biol3:119-125). YAMC cells are often used as an in vitro model for cytokinetreatments of murine IEC (Mei, J. M. et al. (2000) Faseb J 14:1188-1201;Yan, F., and D. B. Polk (2002) J Biol Chem 277:50959-50965). The SV40large T antigen that these cells contained is temperature sensitive, andnon-functional at 37° C. All experiments performed herein involved IFNγtreatment under these non-permissive conditions. In addition, YAMC cellswere serum starved (and IFNγ starved) at 37° C. for 24 hours prior toexperiments. Under such conditions, effects indicating residual Tantigen expression, such as proliferation of cells, were not observed.As a result, effects of IFNγ treatment were due to inherent effects ofIFNγ rather than effects stemming from driving expression of the Tantigen. Furthermore, the upregulation of LY6 family members wasdetected in a second murine cell line, CMT93, confirming that this iseffect is broadly applicable to IEC.

Furthermore, IFNγ was not unique among cytokines for inducing LY6molecules as modest upregulation of LY6 expression was noted aftertreatment with TNFα, IL-1β and, IL-22. The upregulation of LY6 moleculeson IEC in response to IL-22 is interesting in light of recent datademonstrating a potential role for IL-22 in Crohn's Disease (Wolk, K.,et al. J Immunol 178:5973-5981 (2007)). Though homology between mouseand human LY6 molecules are often complicated, there is evidence tosuggest that the upregulation of LY6 molecules is not restricted tomice. Previous studies in rats have suggested upregulation of LY6molecules in the small intestine in colitis models, and it has beensuggested that such expression is involved in inflammation, cell/cellinteractions as well as signaling within the rat IEC (Baksheev, L. etal. J Gastroenterol 41:1041-1052 (2006)).

The data described above indicates that there is a possibility thatlipid raft integrity is involved in LY6C mediated signal transduction inIEC. This implies that disruption of lipid rafts might serve toattenuate downstream affects of LY6C stimulation both by downregulatingLY6C expression and disrupting the structural components of LY6Csignaling. Recently, it has been determined that cholesterol depletionof IEC with statins inhibits proinflamamtory gene expression throughNF-κB modulation (Lee, J. et al., Int Immunopharmacol 7:241-248 (2007)).Furthermore, statins have been effective therapeutics in murine modelsof colitis (Naito, Y., et al. Int J Mol Med 17:997-1004 (2006)). Themechanism linking lipid raft motility and NF-κB blockade remainundetermined, but our data suggests that activation through LY6C couldbe one hypothesis to explain the mechanism of action.

In this study, we identify LY6 molecules as a potential upstream switchin the expression of chemokine genes. Crosslinking of the LY6C receptorwith monoclonal antibodies resulted in dramatic upregulation of nearlyall chemokines analyzed, including CXCL5. We further confirmed thatCXCL5 secretion is greatly enhanced in LY6C crosslinked IEC. It isinteresting that even though both LY6A and LY6C are anchored to the cellsurface by a GPI moiety, and despite higher levels of expression of LY6Athan LY6C on the surface of IEC, that the downstream effects onchemokine secretion are seen with LY6C crosslinking and not consistentlywith LY6A crosslinking.

Example 11 Identification of a Ligand for LYPD5

In this study, a search for ligands of LYPD5 was performed throughtechniques well known to those of ordinary skill in the art, namely byexpression cloning of about 14,000 human genes under CMV promoter intoCOS cells. Pools of 100 genes were transfected into the COS cells grownin 140 wells on 12 well plates. Following transfection, the cells werestained with LYPD5-Fc protein (see FIG. 32). Wells with positivestaining were identified and individual clones were transfected into COScells. A single well expressing a single protein, GLG-1 (ESL-1) wasidentified as a ligand for LYPD5. GLG-1 is characterized by a lengthyextracellular domain (ECD), a transmembrane domain and a cytoplasmicdomain. A series of co-immunoprecipitation studies were conducted usingtechniques known to those of ordinary skill in the art to assess theability of various regions of the GLG-1 ECD to bind LYPD5. It was foundthat variants or fragments of the GLG-1 ECD (see FIGS. 33-35) were ableto serve as a ligand for LYPD5. FIG. 33B shows the results ofco-immunoprecipitation studies using Fragments 1, 2, 3, or 4 as depictedin FIG. 33A and demonstrates that any one of the fragments is sufficientfor LYPD5 binding.

In addition, a GLG-1 ECD domain by itself was found to be sufficient forLYPD5 binding. As shown in FIG. 33, GLG-1 is made up of multiple GLG-1domains and single GLG-1 domains can bind LYPD5. FIG. 34B shows theresults of a co-immunoprecipitation demonstrating that Fragments 1, 2,3, and 4, as well as single GLG-1 domains 115, 150, 215, 538, 609, 670,729, and 858 (as shown in FIG. 34A) were able to bind LYPD5.

Through another co-immunoprecipitation study, binding was shown to bespecific based on fragments of LYPD5 (see FIG. 35A) in which LYPD5 wasfound not to bind BAP negative control, an FN14 negative control was notfound to bind GLG-1 fragment 2, the human GLG-1 domain 115 binds LYPD5,domain 115 is not always expressed at detectable levels but still pullsdown LYPD5, and the fraction of human GLG-1 fragment 1 that lacks domain115 (residues 26-114) does not bind LYPD5 (FIG. 35B).

The “*” in FIGS. 34A and 35A indicates a potential fucosylation site.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

1. A method of treating inflammatory bowel disease (IBD) in a mammal,comprising (i) detecting the level of expression of a nucleic acid orgene encoding a LY6 polypeptide (a) in a test sample of tissue or cellsobtained from said mammal, and (b) in a control sample, wherein a higherlevel of expression of the LY6 nucleic acid or polypeptide in the testsample, as compared to the control sample, is indicative of the presenceof IBD in the mammal from which the test sample was obtained; and (ii)administering to the subject an effective amount of an IBD therapeuticagent.
 2. The method of claim 1, wherein the tissue or cells of the testsample are from the gastrointestinal tract of the mammal.
 3. The methodof claim 2, wherein the tissue or cells of the test sample are from thecolon of the mammal.
 4. The method of claim 1, wherein the controlsample is a sample of normal non-IBD tissue or cells of the same tissueorigin or type, or multiple samples of non-IBD tissue or cells of thesame tissue origin or type the expression levels of which are averaged,or a universal control representing gene expression in multiple samplesof healthy, normal tissue of the same species.
 5. The method of claim 1,wherein the tissue or cells of the test sample are inflamed.
 6. Themethod of claim 1, wherein the tissue or cells of the test sample arenot inflamed.
 7. The method of claim 1, comprising: (a) contacting thetest sample with a detectable agent that specifically binds apolynucleotide that encodes LY6 polypeptide or fragment thereof; (b)contacting the control sample with the detectable agent; and (c)detecting the formation of a complex between the agent and thepolynucleotide of the test sample and the control sample, wherein theformation of less complex in the test sample relative to the controlsample is indicative of the presence of IBD in the mammal.
 8. The methodof claim 7, wherein the polynucleotide comprises the nucleic acidsequence of SEQ ID NO:8, 9, 1, 3, 4, 6, or a fragment thereof comprisingat least 15 contiguous nucleotides of SEQ ID NO:8, 9, 1, 3, 4, or
 6. 9.The method of claim 7, wherein the tissue or cells of the test sampleare from the gastrointestinal tract of the mammal.
 10. The method ofclaim 9, wherein the tissue or cells of the test sample are from thecolon of the mammal.
 11. The method of claim 1, wherein the controlsample is a sample of normal non-IBD tissue or cells of the same tissueorigin or type, or multiple samples of non-IBD tissue or cells of thesame tissue origin or type the expression levels of which are averaged,or a universal control representing gene expression in multiple samplesof healthy, normal tissue of the same species.
 12. The method of claim7, wherein the agent is a second polynucleotide that hybridizes to apolynucleotide having the sequence SEQ ID NO:8, 9, 1, 3, 4, 6 or itscomplement.
 13. The method of claim 12, wherein the secondpolynucleotide comprises a detectable label or attached to a solidsupport.
 14. The method of claim 13, wherein the detectable label isdirectly detectable.
 15. The method of claim 13, wherein the detectablelabel is indirectly detectable.
 16. The method of claim 13, wherein thedetectable label is a fluorescent label.
 17. The method of claim 7,wherein the method is in situ hybridization assay.
 18. The method ofclaim 7, wherein the method is real time polymerase chain reaction(RT-PCR) assay.
 19. The method of claim 1, comprising: (a) contactingthe test sample with a detectable agent that specifically binds a LY6polypeptide or fragment thereof; (b) contacting the control sample withthe detectable agent; and (c) detecting the formation of a complexbetween the agent and the polypeptide of the test sample and the controlsample, wherein the formation of less complex in the test samplerelative to the control sample is indicative of the presence of IBD inthe mammal.
 20. The method of claim 19, wherein the LY6 polypeptidecomprises SEQ ID NO:10, 2, 5, 7 or a fragment thereof comprising atleast 10 contiguous amino acids of SEQ ID NO:10, 2, 5, or
 7. 21. Themethod of claim 19, wherein the tissue or cells of the test sample arefrom the gastrointestinal tract of the mammal.
 22. The method of claim21, wherein the tissue or cells of the test sample are from the colon ofthe mammal.
 23. The method of claim 19, wherein the agent is an antibodyor binding fragment thereof.
 24. The method of claim 23, wherein theantibody or binding fragment thereof comprises a detectable label. 25.The method of claim 24, wherein the detectable label is directlydetectable.
 26. The method of claim 24, wherein the detectable label isindirectly detectable.
 27. The method of claim 24, wherein thedetectable label is a fluorescent label or a radiolabel.
 28. The methodof claim 7 or 19, wherein the tissue or cells of the test sample areinflamed.
 29. The method of claim 7 or 19, wherein the tissue or cellsof the test sample are not inflamed.
 30. The method of claim 1, claim 7or claim 19, wherein the test sample of tissue or cells is obtained froma mammal suspected of experiencing IBD.
 31. The method of claim 1, claim7, or claim 19, wherein the test sample of tissue or cells is obtainedfrom a mammal suspected of experiencing ulcerative colitis (UC).
 32. Themethod of claim 1, claim 7, or claim 19, wherein the tissues or cells ofthe mammal have been contacted with a therapeutic agent, and wherein thelevel of LY6 expression is indicative of the presence or absence of aresponse to the therapeutic agent in the tissue or cells of the mammal.33. The method of claim 1, claim 7, or claim 19, wherein the tissues orcells of the mammal have been contacted with a therapeutic agent,wherein the detecting is a second or subsequent detecting, and whereinthe level of LY6 expression is indicative of the presence or absence ofa response to the therapeutic agent in the tissue or cells of themammal.
 34. The method of claim 1, claim 7, or claim 19, wherein theincrease in LY6 expression in the test sample is at least 1.5 fold, atleast 2 fold, at least 3 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, or at least 10 foldgreater than the control sample.
 35. The method of claim 1, wherein theIBD therapeutic agent is an antagonist of another IBD-associatedmolecule.
 36. The method of claim 35, wherein the IBD-associatedmolecule is a molecule that is differentially expressed in an IBD. 37.The method of claim 36, wherein the IBD-associated molecule isover-expressed in an IBD.
 38. The method of claim 37, wherein theover-expressed IBD-associated molecule is an integrin.
 39. The method ofclaim 38, wherein the IBD-associated molecule is integrin, beta 7(ITGB7).
 40. The method of claim 1, wherein the IBD therapeutic agent isan antagonist of an integrin.
 41. The method of claim 1, wherein the IBDtherapeutic agent is an antagonist of ITGB7.
 42. The method of claim 1,wherein the IBD therapeutic agent is an antagonist of the polypeptideshown as SEQ ID NO:
 69. 43. The method of claim 42, wherein thepolypeptide is encoded by the nucleic acid sequence shown as SEQ ID NO:68.
 44. The method of claim 1, wherein the IBD therapeutic agent is ananti-inflammatory drug.
 45. The method of claim 44, wherein theanti-inflammatory drug is selected from the group consisting ofsulfasalazine and 5-aminosalisylic acid (5-ASA).
 46. The method of claim1, wherein the IBD therapeutic agent is metroidazole.
 47. The method ofclaim 1, wherein the IBD therapeutic agent is ciprofloxacin.
 48. Themethod of claim 1, wherein the IBD therapeutic agent is acorticosteroid.
 49. The method of claim 48, wherein the IBD therapeuticagent is selected from the group consisting of azathioprine and6-mercaptopurine.
 50. The method of claim 1, wherein the IBD therapeuticagent is an anti-diarrheal drug.
 51. The method of claim 1, wherein theIBD therapeutic agent is methotrexate.
 52. The method of claim 1 or 8,wherein the LY6 polypeptide is encoded by a polynucleotide comprisingSEQ ID NO:8.
 53. The method of claim 1 or 8, wherein the LY6 polypeptideis encoded by a polynucleotide comprising SEQ ID NO:9.
 54. The method ofclaim 1 or 8, wherein the LY6 polypeptide is encoded by a polynucleotidecomprising SEQ ID NO:1.
 55. The method of claim 1 or 8, wherein the LY6polypeptide is encoded by a polynucleotide comprising SEQ ID NO:3. 56.The method of claim 1 or 8, wherein the LY6 polypeptide is encoded by apolynucleotide comprising SEQ ID NO:4.
 57. The method of claim 1 or 8,wherein the LY6 polypeptide is encoded by a polynucleotide comprisingSEQ ID NO:6.
 58. The method of claim 1 or 20, wherein the LY6polypeptide comprises amino acid sequence shown as SEQ ID NO:10.
 59. Themethod of claim 1 or 20, wherein the LY6 polypeptide comprises aminoacid sequence shown as SEQ ID NO:2.
 60. The method of claim 1 or 20,wherein the LY6 polypeptide amino acid sequence shown as SEQ ID NO:5.61. The method of claim 1 or 20, wherein the LY6 polypeptide amino acidsequence shown as SEQ ID NO:7.
 62. A method of detecting inflammatorybowel disease (IBD) in a mammal, comprising detecting the level ofexpression of a nucleic acid or gene encoding a LY6 polypeptide (a) in atest sample of inflamed tissue or cells obtained from thegastrointestinal tract of said mammal, and (b) in a control sample,wherein a higher level of expression of the LY6 nucleic acid orpolypeptide in the test sample, as compared to the control sample, isindicative of the presence of IBD in the mammal from which the testsample was obtained.
 63. The method of claim 62, wherein the tissue orcells of the test sample are from the colon of the mammal.
 64. Themethod of claim 62, wherein the control sample is a sample of normalnon-IBD tissue or cells of the same tissue origin or type, or multiplesamples of non-IBD tissue or cells of the same tissue origin or type theexpression levels of which are averaged, or a universal controlrepresenting gene expression in multiple samples of healthy, normaltissue of the same species.
 65. The method of claim 62, comprising: (a)contacting the test sample with a detectable agent that specificallybinds a polynucleotide that encodes LY6 polypeptide or fragment thereof;(b) contacting the control sample with the detectable agent; and (c)detecting the formation of a complex between the agent and thepolynucleotide of the test sample and the control sample, wherein theformation of less complex in the test sample relative to the controlsample is indicative of the presence of IBD in the mammal.
 66. Themethod of claim 65, wherein the polynucleotide comprises the nucleicacid sequence of SEQ ID NO:8, 9, 1, 3, 4, 6, or a fragment thereofcomprising at least 15 contiguous nucleotides of SEQ ID NO:8, 9, 1, 3,4, or
 6. 67. The method of claim 65, wherein the tissue or cells of thetest sample are from the colon of the mammal.
 68. The method of claim62, wherein the control sample is a sample of normal non-IBD tissue orcells of the same tissue origin or type, or multiple samples of non-IBDtissue or cells of the same tissue origin or type the expression levelsof which are averaged, or a universal control representing geneexpression in multiple samples of healthy, normal tissue of the samespecies.
 69. The method of claim 65, wherein the agent is a secondpolynucleotide that hybridizes to a polynucleotide having the sequenceSEQ ID NO:8, 9, 1, 3, 4, 6 or its complement.
 70. The method of claim69, wherein the second polynucleotide comprises a detectable label orattached to a solid support.
 71. The method of claim 70, wherein thedetectable label is directly detectable.
 72. The method of claim 70,wherein the detectable label is indirectly detectable.
 73. The method ofclaim 70, wherein the detectable label is a fluorescent label.
 74. Themethod of claim 65, wherein the method is in situ hybridization assay.75. The method of claim 65, wherein the method is real time polymerasechain reaction (RT-PCR) assay.
 76. The method of claim 62, comprising:(a) contacting the test sample with a detectable agent that specificallybinds a LY6 polypeptide or fragment thereof; (b) contacting the controlsample with the detectable agent; and (c) detecting the formation of acomplex between the agent and the polypeptide of the test sample and thecontrol sample, wherein the formation of less complex in the test samplerelative to the control sample is indicative of the presence of IBD inthe mammal.
 77. The method of claim 76, wherein the LY6 polypeptidecomprises SEQ ID NO:10, 2, 5, 7 or a fragment thereof comprising atleast 10 contiguous amino acids of SEQ ID NO:10, 2, 5, or
 7. 78. Themethod of claim 76, wherein the tissue or cells of the test sample arefrom the colon of the mammal.
 79. The method of claim 76, wherein theagent is an antibody or binding fragment thereof.
 80. The method ofclaim 79, wherein the antibody or binding fragment thereof comprises adetectable label.
 81. The method of claim 80, wherein the detectablelabel is directly detectable.
 82. The method of claim 80, wherein thedetectable label is indirectly detectable.
 83. The method of claim 80,wherein the detectable label is a fluorescent label or a radiolabel. 84.The method of claim 62 or 66, wherein the LY6 polypeptide is encoded bya polynucleotide comprising SEQ ID NO:8.
 85. The method of claim 62 or66, wherein the LY6 polypeptide is encoded by a polynucleotidecomprising SEQ ID NO:9.
 86. The method of claim 62 or 66, wherein theLY6 polypeptide is encoded by a polynucleotide comprising SEQ ID NO:1.87. The method of claim 62 or 66, wherein the LY6 polypeptide is encodedby a polynucleotide comprising SEQ ID NO:3.
 88. The method of claim 62or 66, wherein the LY6 polypeptide is encoded by a polynucleotidecomprising SEQ ID NO:4.
 89. The method of claim 62 or 66, wherein theLY6 polypeptide is encoded by a polynucleotide comprising SEQ ID NO:6.90. The method of claim 62 or 77, wherein the LY6 polypeptide comprisesamino acid sequence shown as SEQ ID NO:10.
 91. The method of claim 62 or77, wherein the LY6 polypeptide comprises amino acid sequence shown asSEQ ID NO:2.
 92. The method of claim 62 or 77, wherein the LY6polypeptide amino acid sequence shown as SEQ ID NO:5.
 93. The method ofclaim 62 or 77, wherein the LY6 polypeptide amino acid sequence shown asSEQ ID NO:7.
 94. The method of claim 62, claim 65 or claim 76, whereinthe test sample of tissue or cells is obtained from a mammal suspectedof experiencing IBD.
 95. The method of claim 62, claim 65 or claim 76,wherein the test sample of tissue or cells is obtained from a mammalsuspected of experiencing ulcerative colitis (UC).
 96. The method ofclaim 62, claim 65 or claim 76, wherein the tissues or cells of themammal have been contacted with a therapeutic agent, and wherein thelevel of LY6 expression is indicative of the presence or absence of aresponse to the therapeutic agent in the tissue or cells of the mammal.97. The method of claim 62, claim 65 or claim 76, wherein the tissues orcells of the mammal have been contacted with a therapeutic agent,wherein the detecting is a second or subsequent detecting, and whereinthe level of LY6 expression is indicative of the presence or absence ofa response to the therapeutic agent in the tissue or cells of themammal.
 98. The method of claim 62, claim 65 or claim 76, wherein theincrease in LY6 expression in the test sample is at least 1.5 fold, atleast 2 fold, at least 3 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, or at least 10 foldgreater than the control sample.